WO2009058690A2 - Nell-1 compositions - Google Patents

Abstract

The present invention provides an angiogenesis or antiangiogenesis composition including a Nell-1 protein and methods of making and using the same.

Description

NELL-I COMPOSITIONS

GOVERNMENT-SPONSORED RESEARCH AND DEVELOPMENT

This work was supported in part by the Wunderman Family Foundation, March of Dimes Birth Defect Foundation (6-FY02-163), NIH/NIDCR RO3 DEO 14649-01,

NIH/NIDCR K23DE00422, NIH DE016107-01, NIH DE016781, the Thomas R. Bales Endowed Chair, the NIH T32 UCLA Research Training Grant, and the UCLA SAIRP NIH- NCI 2U24 CA092865 cooperative agreement. The Government of the United States of America may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention is generally related to a NELL peptide antiangiogenesis or angiogenesis composition and the methods of making and using the same.

BACKGROUND OF THE INVENTION

Angiogenesis is often associated with formation of a tissue where growth of blood vessel is part of the tissue forming process. Promoting angiogenesis can promote formation of a tissue if it is desirable to promote the growth of the tissue, and inhibiting angiogenesis can delay or prevent formation of a tissue if it is desirable to delay or inhibit growth of the tissue. Some examples of the former include tissue repair or bone formation. Some examples of the latter include but are not limited to, delaying or inhibiting growth of a tumor by delaying or inhibiting angiogenesis.

In some other circumstances, angiogenesis can be an undesirable step in formation of a tissue. An example of such a tissue is cartilage. Cartilage is often needed for repairing defects caused by trauma, surgical resection and reconstruction. Unfortunately, current surgical approaches are limited by the availability of donor cartilage, the scarring and morbidity associated with the process, and the rapid degradation of fibrous scars. Tissue engineering techniques, including cells, scaffold and signals, offer an alternative strategy to mediate a more durable repair (TuIi, R., Li, W.J., and Tuan, R.S. 2003. Current state of cartilage tissue engineering. Arthritis Res Ther 5:235-238). The isolation of articular chondrocytes from a small biopsy sample of autologous cartilage, followed by enzymatic isolation, propagation of the cells and reintroduction into a defect after being combined with biodegradable scaffolds, proved its efficacy in recent studies (Gelse, K., and Schneider, H. 2006. Ex vivo gene therapy approaches to cartilage repair. AcIv Drug Deliv Rev 58:259-284). Although the process of harvesting joint cartilage is highly invasive, auricular cartilage is more easily obtained while causing less donor site morbidity. Additionally auricular chondrocytes have a comparable ability to produce new cartilaginous matrix as compared with articular chondrocytes ( Johnson, T. S., Xu, J.W., Zaporojan, V. V., Mesa, J.M., Weinand, C, Randolph, M.A., Bonassar, L. J., Winograd, J.M., and Yaremchuk, M.J. 2004. Integrative repair of cartilage with articular and nonarticular chondrocytes. Tissue Eng 10: 1308-1315).

The primary drawback of harvested chondrocytes is their propensity to dedifferentiate in vitro. As early as one passage in vitro, they lose their chondrocytic phenotype with a decreased ability to secrete proteoglycans and an altered collagen synthesis from type II to type I ( Saraf, A., and Mikos, A.G. 2006. Gene delivery strategies for cartilage tissue engineering. Adv Drug Deliv Rev 58:592-603; Gelse, 2006). The associated risk is chondrocytes which may not produce a cartilage matrix when implanted in vivo ( Benya, P. D., and Shaffer, J.D. 1982. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 30:215-224.). Adult cartilage repair generally remains incomplete, which might be ascribed to the lack of a well-coordinated control system or morphogenetic factors and signaling events (Shapiro, F., Koide, S., and Glimcher, M.J. 1993. Cell origin and differentiation in the repair of full-thickness defects of articular cartilage. J Bone Joint Surg Am 75:532-553). To overcome chondrocyte dedifferentiation, several factors have been introduced to stimulate chondrogenesis, promoting the formation of functionally acceptable cartilage-like repair tissue (Gelse, 2006). Among them, bone morphogenetic proteins (BMPs), insulin-like growth factors (IGFs), fibroblast growth factors (FGFs), and transforming growth factor-β (TGF-β) have been shown to maintain differentiation capacity during chondrocyte expansion. For example, monolayer cultures of rabbit articular chondrocytes were infected with an adenovirus carrying the gene for BMP -2. In these cells BMP -2 greatly increased collagen type II and non-collagenous protein production ( Smith, P., Shuler, F. D., Georgescu, H.I., Ghivizzani, S. C, Johnstone, B., Niyibizi, C, Robbins, P.D., and Evans, CH. 2000. Genetic enhancement of matrix synthesis by articular chondrocytes: comparison of different growth factor genes in the presence and absence of interleukin-1. Arthritis Rheum 43: 1156-1164) and induced the differentiation of chondrocytes ( Grunder, T., Gaissmaier, C, Fritz, J., Stoop, R., Hortschansky, P., Mollenhauer, J., and Aicher, W.K. 2004. Bone morphogenetic protein (BMP)-2 enhances the expression of type II collagen and aggrecan in chondrocytes embedded in alginate beads. Osteoarthritis Cartilage 12:559-567). However, with more time BMP -2 would lead to terminal differentiation including endochondral ossification. This phenomenon is observed as a physiological process in the fetal growth plates, but it can also be observed in growing chondrocytes exposed to BMP-2, and full thickness cartilage defects. Thus, cartilage repair approaches should include stimulation that promotes cartilage formation, but avoids inadvertent effects such as long term bone formation (Gelse, 2006). Growth factors with selective specificity for chondrocytes have yet to be identified. The present invention addresses the above identified problems and needs.

SUMMARY OF THE INVENTION In one aspect, the present invention provides a method of inhibiting angiogenesis in a mammalian subject, comprising administering to the subject an antiangiogenic composition comprising a NELL-I protein, wherein the NELL-I protein is in an amount effective for inhibiting vascular formation in a tissue in the subject. In some embodiments, the NELL-I protein includes a modulated functional domain such that the antiangiogenic effect of the NELL-I is augmented. The modulated domain can be, for example, a TSP-I like domain. In some embodiments, the composition can be effective for cartilage formation. In some embodiments, the composition is effective for antineoplastic application. In some embodiments, the composition is effective for anti-tumor application.

In another aspect, the present invention provides a method of promoting angiogenesis in a mammalian subject, comprising administering to the subject an angiogenic composition comprising a NELL- 1 protein, wherein the NELL- 1 protein includes a modulated functional domain such that the angiogenic effect of the NELL-I is augmented, and wherein the NELL- 1 protein is in an amount effective for promoting vascular formation in a tissue in the subject. In some embodiments, the modulated functional domain is a TSP-I like domain such that the antiangiogenic effect of the TSP-I like domain is negated. In some embodiments, the composition is effective for enhancing bone formation in the tissue.

In a further aspect of the present invention, it is provided an antiangiogenic composition, comprising a NELL-I protein, wherein the NELL- 1 protein includes a modulated functional domain such that the antiangiogenic effect of the NELL-I is augmented. In some embodiments, the composition is effective for cartilage formation. In some embodiments, the composition is effective for antineoplastic application. In some embodiments, the composition is effective for anti-tumor application. In some embodiments, the composition is effective for anti-cancer application. In a further aspect of the present invention, it is provided an angiogenic composition, comprising a NELL-I protein, wherein the NELL-I protein includes a modulated functional domain such that the angiogenic effect of the NELL- 1 is augmented, and wherein the NELL- 1 protein is in an amount effective for promoting vascular formation in a tissue in the subject. In some embodiments, the modulated functional domain is a TSP-I like domain such that the such that the antiangiogenic effect of the TSP-I like domain is negated. In some embodiments, the composition is effective for enhancing bone formation in the tissue.

In the various embodiments above, the mammalian subject can be a patient. BRIEF DESCRIPTION OF THE DRAWINGS Figure IA shows an MOI of 50 pfu/cell produced optimal transfer efficiency three days after AdLacZ transduction, X-gal staining showed that over 80% chondrocytes were stained blue, X200; Figure IB shows western blot probed with antibodies against NeIl-I and β-actin for confirmation of NeIl-I protein expression.

Figure 2 shows the results of AdNeIl-I, AdLacZ or AdBMP -2 transduced chondrocytes 4 weeks after injection into nude mice. AdNeIl-I treated samples had a higher average weight of 23.6mg, 3 times higher than the average of 7.4 mg of AdLacZ treated samples(P<0.05). While AdBMP-2 had the highest weight of 109 mg.

Figures 3A-3C show the results of microCT reconstruction analysis of AdNeIl-I, AdLacZ or AdBMP-2 transduced chondrocytes 4 weeks after injection into nude mice which revealed that the AdBMP-2 samples had significant mineralized area labeled with red color (A). Volume (B) and density (C) were compared among the groups.

Figures 4A-4O show the results of histological analysis of AdNeIl-I, AdLacZ or AdBMP-2 transduced chondrocytes at week 2 after injection into nude mice Chondrocytes nests formed at AdNeIl-I(A) or AdBMP-2 (B) transduced chondrocyte injection sites which were circled by fibroblastic like cells, while AdLacZ (C) treated sites showed mainly fibroblastic like tissue. Alcian blue staining on corresponding tissue sections of Figure 3 A, B, and C to confirm the presence of cartilaginous tissue (blue) (D,E,F). Original magnification for all above figures: 10OX. The expression of CoIX was pronounced detected in hypertrophic chondrocytes in both AdNeIl-I(G) and AdBMP-2(H) groups, but not obviously observed in AdLacZ group(I). p-P38 was more pronounced in the cells nucleus in AdBMP-2 group(K) than in AdNeIl-I(J) or AdLacZ group(L). While p-ERK was a little bit more strongly stained in the chondrocyte nucleus in AdNeIl- 1 group (M) than other two groups(N,O), original magnification X400. original magnification X400. Figures 5A-5M show the results of AdNeIl-I, AdLacZ or AdBMP -2 transduced chondrocytes 4 weeks after injection into nude mice. Samples in AdNeIl-I group showed large area of typical mature cartilage by HE(A) and alcian blue (D). In AdBMP -2 group new bone was formed with blood vessel invasion (B). Small area of cartilage remained demonstrated by alcian blue staining (E). In contrast, AdLacZ samples showed mainly less mature chondrocytes by HE (C) or by alcian blue staining (F), original magnification for above samples XlOO. IHC analysis for NeIl-I in AdNeIl-I group (G), AdBMP-2 group(H) and AdLacZ group(I). IHC analysis for BMP-2 in those groups respectively (J,K,L), and β- galactosidase staining for LacZ(M) were performed. Original magnification X200. Figures 6A-6I show Cbfal(Runx2) which expressed in the nucleus of the cells, was pronounced in AdBMP-2 group (B), but much less obviously observed in AdNeIl-I (A) or AdLacZ group (C), original magnification X400. CoIX was intensively stained in hypertrophic chondrocytes in AdBMP-2 (E) or AdNeIl-I groups (D) and in the extracellular matrix of AdBMP-2 groups (E). The staining was less expressed in the chondrocytes in AdLacZ group (F). Another chondrogenic marker, the expression patterns of Tenascin X, was pronounced detected in hypertrophic chondrocytes in AdNeIl- 1 (G), which was only weakly detected in the extracellular matrix in AdBMP-2 group(H) and not detectable in AdLacZ group(I). Original magnification X200.

Figures 7A-7F show that AdBMP-2 samples (B) had strong expression of VEGF which was not detectable in those AdNeIl-I and AdLacZ groups (A, C). And the osteogenic marker osteocalcin (OCN) was also strongly stained only in the extracellular matrix from AdBMP-2 group (E) and not detectable in the other two groups (D,F). Original magnification X200.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a composition comprising a NELL-I protein and a method of using the composition. The composition includes a NELL-I protein in a form and/or amount effective for applications as described below.

In some embodiments, the antiangiogenic effects of a NELL-I protein can be modulated, e.g., by modulating functional sites on the NeIl-I molecule. As used herein, the term "functional side" refers to a site or domain on the NELL-I that imparts an antiangiogenesis effect to the molecule. For example, one such side of the NELL-I protein is the TSP-I like domain on the NELL-I molecule. In some embodiments, modulating the functional sites on the NELL-I molecule includes, e.g., mutating the TSP-I like domain to negate its effects to improve angiogenesis. Compositions or methods of using the composition containing a NELL- 1 thus modulated are effective for bone applications, which are described below. In these embodiments, the composition can optionally include a bioactive agent or material that is angiogenic. Such bioactive agents are, for example, hyauronic acid (HA), or a bone morphogenetic protein (BMP).

In some embodiments, modulating the functional sites on the NELL-I molecule includes, e.g., mutating the TSP-I like domain to augment its effects to prevent angiogenesis. Compositions or methods of using the composition containing a NELL- 1 thus modulated are effective for any application pertaining or related to antiangiogenesis. Some examples of such applications include, but are not limited to, antineoplastic application, and cartilage applications, which are described below.

In some embodiments, the present invention provides a composition and method of using the composition for inhibiting vascular endothelial growth factor (VEGF) expression in a mammalian subject by administering to the subject a composition comprising a NELL-I protein. VEGF expression is a necessary step in angiogenesis. In these embodiments, the NELL- 1 protein is effective as an antiangiogenic agent or an antineoplastic agent.

The composition can be in a formulation for systemic or local delivery/administration. Formulations suitable for systemic or local administration are described below.

Definitions

The term "antibody", as used herein, includes various forms of modified or altered antibodies, such as an intact immunoglobulin, an Fv fragment containing only the light and heavy chain variable regions, an Fv fragment linked by a disulfide bond (Brinkmann et al. (1993) Proc. Natl. Acad. Sci. USA, 90: 547-551), an Fab or (Fab)'2 fragment containing the variable regions and parts of the constant regions, a single-chain antibody and the like (Bird et al. (1988) Science 242: 424-426; Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85: 5879- 5883). The antibody can be of animal (especially mouse or rat) or human origin or can be chimeric (Morrison et al. (1984) Proc Nat. Acad. Sci. USA 81 : 6851-6855) or humanized (Jones et al. (1986) Nature 321 : 522-525, and published UK patent application #8707252).

The terms "binding partner", or "capture agent", or a member of a "binding pair" refers to molecules that specifically bind other molecules to form a binding complex such as antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, etc. The terms "carrier," "pharmaceutically acceptable carrier," "delivery vehicle," or "vehicle" can be used interchangeably.

The term "specifically binds", as used herein, when referring to a biomolecule (e.g. protein, nucleic acid, antibody, etc.), refers to a binding reaction which is determinative of the presence biomolecule in heterogeneous population of molecules (e.g. proteins and other biologies). Thus, under designated conditions (e.g. immunoassay conditions in the case of an antibody or stringent hybridization conditions in the case of a nucleic acid), the specified ligand or antibody binds to its particular "target" molecule and does not bind in a significant amount to other molecules present in the sample. The term osteoporosis refers to a heterogeneous group of disorders characterized by decreased bone mass and fractures. Clinically, osteoporosis is segregated into type I and type II. Type I osteoporosis occurs predominantly in middle aged women and is associated with estrogen loss at the menopause, while osteoporosis type II is associated with advancing age.

Osteogenesis imperfecta (OI) refers to a group of inherited connective tissue diseases characterized by bone and soft connective tissue fragility (Byers & Steiner (1992) Annu. Rev. Med. 43: 269-289; Prockop (1990) J. Biol. Chem. 265: 15349-15352). Males and females are affected equally, and the overall incidence is currently estimated to be 1 in 5,000-14,000 live births. Hearing loss, dentinogenesis imperfecta, respiratory insufficiency, severe scoliosis and emphysema are just some of the conditions that are associated with one or more types of OL While accurate estimates of the health care costs are not available, the morbidity and mortality associated with OI certainly result from the extreme propensity to fracture (OI types I-IV) and the deformation of abnormal bone following fracture repair (OI types II-IV).

The term "osteogenic cells" refers to cells capable of mineralizing. Osteogenic cells include osteoblasts, osteoblast like cells, mesenchymal cells, fibroblast cells, fetal embryonic cells, stem cells, bone marrow cells, dura cells, chrondrocytes, and chondroblastic cells.

The term "osteochondroprogenitor" refers to any cell capable of forming cartilage, e.g., less differentiated osteogenic cells which are capable of mineralizing and/or forming cartilage. Osteochondroprogenitor cells include osteoblasts, osteoblast like cells, mesenchymal cells, fibroblast cells, fetal embryonic cells, stem cells, bone marrow cells, dura cells, chrondrocytes, and chondroblastic cells.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

The term "animal" refers to a mammal, which includes any warm-blooded vertebrate of the class Mammalia, whose females possess milk-secreting mammae for the nourishment of the young, which includes for example: human beings, horses, dogs, cats, rodents, cattle, whales, bats, etc. The term "protein structure" can refer to protein structure forms derived experimentally or through computer assisted soft ware predictions. Common experimental methods used to determine a protein's structure are x-ray crystallography and nuclear magnetic resonance (NMR). In x-ray crystallography, scientists determine protein structure by measuring the directions and intensities of x-ray beams diffracted from high-quality crystals of a purified protein molecule. NMR uses high magnetic fields and radio-frequency pulses to manipulate the spin states of nuclei. The positions and intensities of the peaks on the resulting spectrum reflect the chemical environment and nucleic positions within the molecule. The term "domain" can refer to any discrete portion of the NELL-I molecule that has an already defined function, an anticipated function, or a function to be defined in the future. Examples of anticipated NELL-I domains with anticipated functions are described in U.S. application No. 11/713,366. filed on March 1, 2007, the teaching of which is incorporated herein in its entirety by reference. For example, the observed strong binding interaction between recombinant NeIl-I and heparin sulfate may possibly be mediated by TSP-N

(Kuroda, S., M. Oyasu, et al. (1999). "Biochemical characterization and expression analysis of neural thrombospondin- 1 -like proteins NELL-I and NELL2." Biochem Biophys Res Commun 265(1): 79-86.). The TSP-N module in NeIl-I can potentially interact with cell surface heparin sulfate proteoglycans to mediate general cellular functions such as spreading, focal adhesion, disassembly, and endocytosis (Bornstein, P. (1995). "Diversity of function is inherent in matricellular proteins: an appraisal of thrombospondin 1." J Cell Biol 130(3): 503- 6.).

Additionally, NELL-I may be a member of the chordin-like CR domain family, which includes chordin, kielin, crossveinless, twisted gastrulation, and connective-tissue growth factor (Abreu, J. G., N. I. Ketpura, et al. (2002). "Connective-tissue growth factor

(CTGF) modulates cell signaling by BMP and TGF-beta." Nat Cell Biol 4(8): 599-604). CR domains can mediate specific interactions with BMPs and other members of the TGF-β superfamily in either a pro- or anti-ligand fashion (Abreu, Ketpura et al. 2002). In addition, CR domains affect other functions such as receptor binding and trimer formation. However, it is conceivable that new domains could be identified in NELL-I and the definition herein shall not be limiting. Overall it is anticipated that NELL- 1 will have specific domains that carry out specific functions such as protein secretion, ligand binding, trimer or tetramer formation etc.

The term "conformational change" refers to any change in NELL- 1 protein structure as a result of microenvironmental interactions such as ionic interactions, hydrophobic/hydrophilic interactions, protein interactions, receptor interactions, cell-cell interactions, etc. For example, heparin sulfate binding to NELL-I is known to induce a conformational change based on differential anti-NELL- 1 antibody binding characteristics in the presence or absence of heparin sulfate.

The term "cartilage" refers to all forms of cartilage including, but not limited to, hyaline, elastic, and fibrocartilage.

The term proteoglycan can refer to various extracellular matrix molecules including heparin sulfate, heparan sulfate, dermatan sulfate, chondroitin sulfate.

The term glycosaminoglycan can refer to various extracellular matrix molecules including hyaluronic acid.

The term heparin and heparan sulphate refer to molecules that both have the same basic structure consisting of repeating disaccharides of GIcUA and GIcNAc. The size of an individual chain can reach 100 kDa, but normally they are below 50 kDa. Heparin is widely known for its anti-coagulant action, the one based on its binding with antithrombin III. Distinction between heparin and heparan sulphate is difficult, since both structural and functional criteria are inadequate to separate these two forms. They both contain numerous variations of sulphation and L-epimerization. N-deacetylation and the successive N-sulfation appear to be the critical steps, since the additional modifications locate mainly in the regions where N-sulfation has already occurred. The amount of N-sulfation has occasionally been used to make distinction between heparin and heparan sulphate so that in heparan sulphate the proportion of N-sulfation is below 50% (Fraansson, L. A., I. Carlstedt, et al. (1986). "The functions of the heparan sulphate proteoglycans." Ciba Found Symp. 124: 125-42), while in heparin it is usually 70% or higher (Roden, L., S. Ananth, et al. (1992). "Heparin— an introduction." Adv Exp Med Biol, 313: 1-20). Epimerization of the glucuronic acid and the successive 2-sulphation are typical for both glycosaminoglycans. Sulphation at position 2 of glucuronic acid seems to prevent the epimerization backwards to the form that is more favored energetically.

The term "effective for angiogenesis" refers to angiogenesis in a tissue enhanced by a factor of 0.5% or above, for example, by from about 0.5% to about 1000 x 100%, by from about 0.5% to about 100 x 100%, or by from about 0.5% to about 50 x 100%. In some embodiments, the term "effective for angiogenesis" can refer to angiogenesis in a tissue ehnahced by a factor of about 1%, about 2%, about 5%, about 10%, about 20%, about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%. The term "effective for antiangiogenesis" refers to angiogenesis in a tissue reduced by a factor of 0.5% or above, for example, by from about 0.5% to about 1000 x 100%, by from about 0.5% to about 100 x 100%, or by from about 0.5% to about 50 x 100%. In some embodiments, the term "effective for antiangiogenesis" can refer to angiogenesis in a tissue ehnahced by a factor of about 1%, about 2%, about 5%, about 10%, about 20%, about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%. In some further embodiments, the term "effective for antiangiogenesis" can refer to complete inhibition of angiogenesis in a tissue.

The term "effective for antitumor" refers to an antitumor effect where growth of the tumor is delayed by a factor of 0.5% or above, for example, by from about 0.5% to about

1000 x 100%, by from about 0.5% to about 100 x 100%, or by from about 0.5% to about 50 x 100%. In some embodiments, the term "effective for antitumor" refers to an antitumor effect where growth of the tumor is delayed by a factor of about 1%, about 2%, about 5%, about 10%, about 20%, about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%. In some further embodiments, the term "effective for antitumor" refers to an antitumor effect where growth of the tumor is completely stopped. Alternatively, in some embodiments, the term "effective for antitumor" refers to an antitumor effect where the size of the tumor is reduced by a factor of 0.5% or above, for example, by from about 0.5% to about 1000 x 100%, by from about 0.5% to about 100 x 100%, or by from about 0.5% to about 50 x 100%. In some embodiments, the term "effective for antitumor" refers to an antitumor effect where the size of the tumor is reduced by a factor of about 1%, about 2%, about 5%, about 10%, about 20%, about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, or about 900%. In some further embodiments, the term "effective for antitumor" refers to an antitumor effect where the tumor is completely disappear.

As used herein, the term effective for inhibiting vascular formation in a tissue refers to vascular formation, as compared with vascular formation in a subject without receiving enhanced exposure to NELL, delayed or reduced by at least 5% to 100%, by at least 10% to 100%, by at least 20% to 100%, by at least 30% to 100%, by at least 40% to 100%, by at least 50% to 100%, by at least 60% to 100%, by at least 70% to 100%, by at least 80% to 100%, by at least 90% to 100%, by at least 95% to 100%, or by at least 99%. As used herein, the term antiangiogenic effect of NELL-I refers to delaying or reducing angiogenesis in a tissue receiving NELL-I, as compared with a tissue without enhanced or increased exposure to NELL-I, by at least 5% to 100%, by at least 10% to 100%, by at least 20% to 100%, by at least 30% to 100%, by at least 40% to 100%, by at least 50% to 100%, by at least 60% to 100%, by at least 70% to 100%, by at least 80% to 100%, by at least 90% to 100%, by at least 95% to 100%, or by at least 99%. The term antiangiogenic effect of the NELL-I is augmented refers to the antiangiogenic effect of NELL-I peptide, as compared with the antiangiogenic effect of NELL-I without domain modification, increased by at least 5% or higher (e.g., by 5% to 2000% or to 10000%), at least 10% or higher, by at least 20% or higher, by at least 30% or higher, by at least 40% or higher, by at least 50% or higher, by at least 60% or higher, by at least 70% or higher, by at least 80% or higher, by at least 90% or higher, or by at least 100% or higher.

As used herein, the term angiogenic effect of NELL-I refers to increasing angiogenesis in a tissue receiving NELL-I, as compared with a tissue without enhanced or increased exposure to NELL-I, by at least 5% or higher (e.g., by 5% to 2000% or to 10000%), at least 10% or higher, by at least 20% or higher, by at least 30% or higher, by at least 40% or higher, by at least 50% or higher, by at least 60% or higher, by at least 70% or higher, by at least 80% or higher, by at least 90% or higher, or by at least 100% or higher. The term angiogenic effect of the NELL-I is augmented refers to angiogenic effect of NELL-I peptide, as compared with the angiogenic effect of NELL-I without domain modification, increased by at least 5% or higher (e.g., by 5% to 2000% or to 10000%), at least 10% or higher, by at least 20% or higher, by at least 30% or higher, by at least 40% or higher, by at least 50% or higher, by at least 60% or higher, by at least 70% or higher, by at least 80% or higher, by at least 90% or higher, or by at least 100% or higher. As used herein, the term a TSP-I like domain refers to a protein domain functionally and/or structurally similar to or equivalent of a TSP-I like domain. The term "similar" shall mean a substantial similarity in functionality and/or structure.

As used herein, the term effective for cartilage formation refers to increased formation of cartilage, as compared with cartilage formation in a subject without receiving enhanced exposure to NELL-I, by at least 5% or higher (e.g., by 5% to 2000% or to 10000%), at least 10% or higher, by at least 20% or higher, by at least 30% or higher, by at least 40% or higher, by at least 50% or higher, by at least 60% or higher, by at least 70% or higher, by at least 80% or higher, by at least 90% or higher, or by at least 100% or higher. As used herein, the term effective for bone formation refers to increased formation of bone, as compared with bone formation in a subject without receiving enhanced exposure to NELL-I, by at least 5% or higher (e.g., by 5% to 2000% or to 10000%), at least 10% or higher, by at least 20% or higher, by at least 30% or higher, by at least 40% or higher, by at least 50% or higher, by at least 60% or higher, by at least 70% or higher, by at least 80% or higher, by at least 90% or higher, or by at least 100% or higher.

As used herein, the term effective for antineoplastic application refers to delaying or reducing abnormal growth of cells, which may lead to a neoplasm or tumor, as compared with abnormal growth of cells in a subject without receiving enhanced exposure to NELL, by at least 5% to 100%, by at least 10% to 100%, by at least 20% to 100%, by at least 30% to 100%, by at least 40% to 100%, by at least 50% to 100%, by at least 60% to 100%, by at least 70% to 100%, by at least 80% to 100%, by at least 90% to 100%, by at least 95% to 100%, or by at least 99%.

As used herein, the term effective for anti-tumor application refers to delaying or reducing tumor growth, as compared with tumor growth in a subject without receiving enhanced exposure to NELL, by at least 5% to 100%, by at least 10% to 100%, by at least 20% to 100%, by at least 30% to 100%, by at least 40% to 100%, by at least 50% to 100%, by at least 60% to 100%, by at least 70% to 100%, by at least 80% to 100%, by at least 90% to 100%, by at least 95% to 100%, or by at least 99%.

As used herein, the term effective for anti-tumor application refers to delaying or reducing cancer growth, as compared with cancer growth in a subject without receiving enhanced exposure to NELL, by at least 5% to 100%, by at least 10% to 100%, by at least 20% to 100%, by at least 30% to 100%, by at least 40% to 100%, by at least 50% to 100%, by at least 60% to 100%, by at least 70% to 100%, by at least 80% to 100%, by at least 90% to 100%, by at least 95% to 100%, or by at least 99%.

NELL-I peptide

NELL-I peptide is an 810 amino acid peptide, distributed primarily in bone. NELL-I peptide is a trimeric peptide and has an amino sequence as reported by Ting (Ting et al. (1999) J Bone Mineral Res, 14: 80-89; and GenBank Accession Number U57523)). In some embodiments, a NELL-I protein is a protein expressed by the NELL-I gene or cDNA (SEQ ID NO: 1, 2 and 3), which is disclosed by Watanabe et al. (1996) Genomics 38 (3): 273-276; Ting et al. (1999) J Bone Mineral Res, 14: 80-89; and GenBank Accession Number U57523), and includes SEQ ID NO: 2, 4, and 6. The NELL-I protein can include NELL-I protein fragments that retain the ability to induce bone mineralization. The NELL-I protein can be a native NELL-I protein or a recombinant protein. The term "NELL-I" protein includes NELL-I peptide, a fragment thereof, or a derivative thereof. The term NELL-I protein also includes functional equivalents or conformational equivalents of NELL-I peptide. Functional equivalents or conformational equivalents of NELL-I can be derived by reference to functional domain structures or conformational structures of NELL-I . TSP-I like domain

NeIl-I has many functional domains. For example, it has a partial TSP-I -like domain. Thrombospondin (TSP)-I (SEQ ID NO: 7) is an antiangiogenic extracellular matrix glycoprotein that modulates several aspects of cellular function. This is consistent with the observation that NeIl-I overexpression down regulates VEGF (see, e.g., descriptions in U.S. Patent No. 7,052,856, the teaching of which is incorporated herein in its entirety by reference).

Modulation of a NELL-I protein by modulating a functional side/domain such as TSP-I can include, for example, mutating the functional side to negate or enhance the function of the functional side. Such modulating can be chemical modulation or biochemical modulation. Chemical modulation can be, e.g., PEGylation or methylation, which are well documented in the art. Biochemical modulation can be manipulation of a site in the NELL-I gene or cDNA that expresses the functional side/domain. For example, the site in the NELL- 1 gene or cDNA that expresses the TSP-I like domain can be partially or fully knocked out such that the NELL-I gene or cDNA will express a NELL- 1 protein that includes no or only partial sequence of the TSP-I like domain. In some embodiments, a gene or nucleic acid construct expressing one or more TSP-I like domains can be inserted in The NELL-I gene or cDNA to express a NELL-I protein that includes one or more than one full TSP-I like domains. Such a NELL-I peptide can have an augmented angiogenic effect.

Methods of chemical or biochemical modulation of a protein or a gene/DNA expressing the protein are well documented in the art. Some general references describing methods of chemical and/or biochemical modulation of a protein and/or a gene/DNA expressing the protein are, e.g., Cell Engineering, Mohamed Al-Rubeai, Ed., Spinger, 1999; and Wang, L. H., et al, Nature Medicine, 10:40-47 (2004)).

NELL-I Related Agents

In some other embodiments, the term "NELL-I" peptide or protein can be a NELL-I related agent, which can be a fragment of NELL-I peptide, a derivative of NELL-I peptide, a splice variant of NELL-I peptide, or a structural, functional, or conformational equivalent of NELL-I peptide.

Computer structural simulation of NELL-I has been reported. The peptide is reported to have a structure as shown in Figures 4A-4D. Critical functional domains of NELL-I include but are not limited to the regions shown in Figures 2, 3, and 4A-4D.

Accordingly, in one embodiment, the NELL-I related agent can be a peptide or protein that has one or more function domains of NELL-I, as described above, or a functional equivalent of any of or a combination of these functional domains. In some embodiments, the functional domains can include mutated sequences and/or sequence knocked-outs provided that the domains function remain substantially unchanged.

In some embodiments, the NELL-I related agent can be can be a peptide or polypeptide having an degree of homology of about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 99% to a NELL-I protein. In some embodiments, the NELL-I related agent can be a conformational equivalent of any or all the functional domains of NELL-I peptide. Such conformational equivalent(s) can have an amino acid sequence similar to that of NELL, e.g., having a degree of homology of about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 99% to NELL-I protein. In some other embodiments, the conformational equivalent can have an amino acid sequence substantially different or unrelated to NELL-I peptide, provided that such equivalent(s) have a 2D or 3D conformation substantially similar to the 2D or 3D conformation of any or all the functional domains of NELL-I peptide or substantially similar to the 2D or 3D conformation of NELL-I peptide, one of which is shown in Figure 2. The 2D or 3D conformation can be, but not limited to, NELL-I protein structure forms derived experimentally or through computer assisted soft ware predictions. Although not yet described for NELL-I, examples of a conformational equivalent can have substantially different amino acid sequences include the example of bone morphogenetic protein 7 and growth differentiation factor 5 (Schreuder et al. Crystal structure of recombinant human growth and differentiation factor 5: Evidence for interaction of the type I and type II receptor-binding sites. Biochemical and Biophysical Research Communications 329 (2005) 1076-1086).

In some embodiments, the NELL-I related agent can be a compound whose primary protein structure is different from that of NELL-I but has a final structure that is similar or the same as that of NELL-I.

In some further embodiments, the NELL-I related agent also includes splice variants of NELL-I peptide. Exons in the NELL-I peptide can be knocked out so as to make splice variants of NELL-I peptide. For example, NELL-I can be spliced into two or three fragments forming the trimeric NELL-I peptide by splicing NELL-I along the two exon regions. Methods and procedures for making splice variants of a protein or peptide are well known in the art (see, U.S. application publication No. 20050148511, the teaching of which is incorporated herein by reference).

In some embodiments, the NELL-I peptide described herein can be a derivative of the NELL-I peptide. The term "derivative" as used herein, refers to any chemical or biological compounds or materials derived from a NELL- 1 peptide, structural equivalents thereof, or conformational equivalents thereof. For example, such a derivative can include any pro-drug form, PEGylated form, or any other form of a NELL- 1 peptide that renders the NELL- 1 peptide more stable or to have a better osteophilicity or lipophilicity. In some embodiments, the derivative can be a NELL-I peptide attached to poly(ethylene glycol), a poly(amino acid), a hydrocarbyl short chain having C1-C20 carbons, or a biocompatible polymer. In some embodiments, the term "derivative" can include a NELL-I peptide mimetics. Synthesis of mimetics of a peptide is well document in the art. The following describes an example of the basic procedure for the synthesis of a peptide, including a peptide mimetics: Before the peptide synthesis starts, the amine terminus of the amino acid (starting material) can protected with FMOC (9-fluoromethyl carbamate) or other protective groups, and a solid support such as a Merrifield resin (free amines) is used as an initiator. Then, step (1) through step (3) reactions are performed and repeated until the desired peptide is obtained: (1) a free-amine is reacted with carboxyl terminus using carbodiimide chemistry, (2) the amino acid sequence is purified, and (3) the protecting group, e.g., the FMOC protecting group, is removed under mildly acidic conditions to yield a free amine. The peptide can then be cleaved from the resin to yield a free standing peptide or peptide mimetics. In some embodiments, the peptide derivative described herein includes a physically or chemically modified NELL-I peptide. Physically modified peptide can be modification by, for example, modification by ionic force such as forming an ionic pair with a counterion, modification by hydrogen bonding, modification by modulation of pH, modulation by solvent selection, or modification by using different protein folding/unfolding procedures, which can involve selection of folding/unfolding temperature, pH, solvent, and duration at different stage of folding/unfolding.

In some embodiments, the peptide derivative can include a chemically modified NELL-I peptide. For example, a short hydrocarbon group(s) (e.g. methyl or ethyl) can be selectively attached to one or multiple sites on the NELL-I peptide molecule to modify the chemical and/or physical properties of the peptide. In some embodiments, a mono-, oligo- or poly(ethylene glycol) (PEG) group(s) can be selectively attached to one or multiple sites on the NELL-I peptide molecule to modify the chemical and/or physical properties of the peptide by commonly known protein PEGylation procedures (see, e.g., Mok, H., et al., MoI. Then, l l(l):66-79 (2005)). INHIBITION OF VEGF Expression BY NELL-I

One of the most specific and critical regulators of angiogenesis is vascular endothelial growth factor (VEGF), which regulates endothelial proliferation, permeability, and survival. Substantial evidence also implicates VEGF as an angiogenic mediator in tumors and intraocular neovascular syndromes (Pandya, N.M., Dhalla, N.S., and Santani, D. D. 2006. Angiogenesis— a new target for future therapy. Vascul Pharmacol 44:265-274) Angiogenesis, the growth of new blood vessels, is essential during tissue repair, foetal development, and female reproductive cycle. In contrast, uncontrolled angiogenesis promotes tumor and retinopathies, while inadequate angiogenesis can lead to coronary artery disease. A balance between pro-angiogenic and antiangiogenic growth factors and cytokines tightly controls angiogenesis. With the identification of several proangiogenic molecules such as the vascular endothelial cell growth factor (VEGF), the fibroblast growth factors (FGFs), and the angiopoietins, and the recent description of specific inhibitors of angiogenesis such as platelet factor-4, angiostatin, endostatin, and vasostatin, it is recognized that therapeutic interference with vasculature formation offers a tool for clinical applications in various pathologies. Inhibition of angiogenesis can prevent diseases such as cancer, diabetic nephropathy, arthritis, psoriasis, and possibly various forms of arthritis whereas stimulation of angiogenesis is beneficial in the treatment of coronary artery disease (CAD), cardiac failure, tissue injury, etc. (Pandya, 2006).

VEGF is an essential coordinator of chondrocyte death, chondroclast function, extracellular matrix remodeling, angiogenesis and bone formation in the growth plate. Inhibiting VEGF by a soluble receptor chimeric protein (FIt-(I -3)-IgG) resulted in almost complete suppression of blood vessel invasion, impaired trabecular bone formation, and impaired expansion of hypertrophic chondrocyte zone. Recruitment and/or differentiation of chondroclasts, which express gelatinase B/matrix metalloproteinase-9, and resorption of terminal chondrocytes also decreased (Gerber, H.P., Vu, T. H., Ryan, A.M., Kowalski, J., Werb, Z., and Ferrara, N. 1999. VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat Med 5:623-628). Surprisingly, in this application we show that NELL- 1 is able to stimulate chondrocyte hypertrophy in the absence of VEGF upregulation. This implies that angiogenesis and chondrocyte hypertrophy may not necessarily be coupled and that chondrocyte hypertrophy can occur in the absence of mineralization and/or bone formation. Bone growth can occur by endochondral ossification or intramembranous ossification and VEGF appears to be important for both (VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat Med 5:623-628; Skeletal defects in VEGF(120/120) mice reveal multiple roles for VEGF in skeletogenesis. Development 129: 1893-1904). Cbfal/Runx2 is expressed in osteoblasts, prechondrogenic mesenchymal condensations and hypertrophic chondrocytes and it is an indispensable element for a tissue-specific genetic program that regulates VEGF expression during endochondral ossification. VEGF has been found to be essential for hypertrophic chondrocyte apoptosis and angiogenesis in the mandibular condyle, indicating that it plays a central role in endochondral ossification (Li, Q.F., and Rabie, A.B. 2007. A new approach to control condylar growth by regulating angiogenesis. Arch Oral Biol 52: 1009-1017). Vascular invasion is prerequisite for bone formation. Osteogenesis, which is the formation of the new bone, and angiogenesis, which is the invasion of new blood vessels, are closely related processes. Growth of cartilage canals into the secondary ossification centre was improved by VEGF. The cartilage canals contain chondroclasts which opened the lacunae of hypertrophic chondrocytes.

Vascularization is a crucial event in endochondral ossification. Vascularization allows the invasion of mesenchymal cells into the empty lacunae of hypertrophic chondrocytes and formation of an osteoid layer. The accelerated cartilage resorption was possibly due to new blood vessel invasion, which leads to the recruitment of chondroblasts into the area of bone formation. These two processes may act together to enhance bone formation. Thus, VEGF- dependent new blood vessel recruitment is essential for coupling cartilage resorption and mineralized bone formation during the process of endochondral ossification in bone development. Blood vessel invasion of cartilage, which is normally avascular, is the first crucial step in this process_(Li, 2007). Thus the surprising ability of NELL-I to uncouple chondrocyte hypertrophy from VEGF upregulation allows unprecedented control over bone and cartilage formation. In situations where more bone (and more angiogenesis) is desired, the inhibitory effects of the NELL-I molecule on angiogenesis can be negated by modifications to the NELL-I molecule or inhibitors to certain antiangiogenic domains of the NELL- 1 molecule. In situations where more cartilage formation is desired, the inhibitory effects of the NELL-I molecule on angiogenesis can be augmented by modifications to the NELL- 1 molecule or or enahncers to certain antiangiogenic domains of the NELL- 1 molecule. Administration of exogenously produced NELL-I The NELL-I proteins, or biologically active fragments thereof, of this invention are useful for intravenous, parenteral, topical, oral, or local administration (e.g., by aerosol or transdermally). Exemplary modes of administration include intra-arterial injection, injection into fracture sites, and delivery in a biodegradable matrix. The NELL-I protein agents are typically combined with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition. Pharmaceutically acceptable carriers can contain a physiologically acceptable compound that acts, e.g., to stabilize the composition or to increase or decrease the absorption of the agent. Physiologically acceptable compounds can include, e.g., carbohydrates (e.g., glucose, sucrose, and dextrans), antioxidants (e.g., ascorbic acid and glutathione), chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the anti-mitotic agents, excipients, and other stabilizers and/or buffers.

Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents and preservatives that are useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, e.g., phenol and ascorbic acid. One skilled in the art would appreciate that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, e.g., on the rout of administration of the anti-mitotic agent and on the particular physio-chemical characteristics of the anti-mitotic agent. Some formulations for the delivery of bone morphogenic proteins (BMPs) are described in detail in U.S. Pat. 5,385,887.

The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include powder, tablets, pills, capsules and lozenges. It is recognized that the NELL- 1 protein(s), if administered orally, should be protected from digestion. This is typically accomplished either by complexing the protein with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the protein in an appropriately resistant carrier such as a liposome. Means of protecting compounds from digestion are well known in the art (see, e.g., U.S. Pat. 5,391,377 describing lipid compositions for oral delivery of therapeutic agents).

The pharmaceutical compositions of this invention are useful for topical administration, e.g., in surgical wounds to facilitate bone reconstruction and/or repair. In another embodiment, the compositions are useful for parenteral administration, such as intravenous administration or administration into a body cavity or lumen of an organ. The compositions for administration will commonly comprise a solution of the NELL-I protein dissolved in a pharmaceutically acceptable carrier, e.g., an aqueous carrier for water-soluble proteins. A variety of carriers can be used, e.g., buffered saline and the like. These solutions should be sterile and free of undesirable matter. These compositions can be sterilized by conventional sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, toxicity-adjusting agents and the like, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.

The concentration of NELL-I proteins in these formulations can vary widely, and can be selected based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs. In one embodiment, the NELL- 1 proteins are utilized in the form of a pharmaceutically acceptable solution (including reconstitution from a lyophilized form). In certain embodiments, the NELL-I proteins are solubilized at concentrations of at least about 1 mg/ml, or about 2 to 8 mg/ml, so that a pharmaceutically effective amount of protein can be delivered without undue volumes of carrier being necessary. For some applications, concentrations above 2 mg/ml may be desirable.

As alluded to above, the dosage regimen will be determined by the clinical indication being addressed, as well as by various patient variables (e.g., weight, age, sex) and clinical presentation (e.g., extent of injury, site of injury, etc.). In certain embodiments, the dosage of NELL-I proteins is in the range from about 1 to about 10000 μg, or from about 10 to 1000 μg, or from about 10 to 100 μg. Graft materials Bone wounds, as well as many other wound models, initiate a release of biologically active agents critical to the wound healing process. Bone morphogenic proteins (BMPs), which naturally occur in bone, once released from the wound, stimulate osteoinduction and regenerate lost or damaged bone tissue. These same proteins, in a purified form, can be used to stimulate bone growth into a biodegradable matrix, allowing for artificial creation of bone both within and external to the normal skeletal boundaries. NELL-I proteins can be used to stimulate bone re-mineralization in a manner analogous to the use of bone morphogenic proteins.

NELL-I proteins can be administered systemically as discussed above. In addition, or alternatively, the NELL- 1 proteins can be applied directly to a bone or bone fracture site. This can be accomplished by direct injection or during surgery (e.g., when setting complex fractures, when reconstructing bone, when performing bone transplants, etc.).

In certain embodiments, e.g., where bone reconstruction or repair is performed surgically, the NELL-I protein can be administered using a sustained delivery "vehicle". Sustained delivery vehicles include, but are not limited to, biodegradable delivery vehicles. In some embodiments, biodegradable delivery vehicles are porous.

Biodegradable porous delivery vehicles have been developed for the controlled release of substances while also providing a location for cellular attachment and guided tissue regeneration. Biodegradable materials can be categorized as: 1) those that are hydrophilic, and 2) those that are hydrophobic. Hydrophilic materials (e.g., demineralized freeze-dried bone, ceramic, fibrin, gelatin, etc.) possess a high affinity for water, which provides for ready incorporation of aqueous NELL-I protein solutions within the internal porosity of the material. Hydrophobic materials (e.g., poly(L-lactic acid), poly(D,L-lactic acid), poly(glycolic acid), etc.), while potentially broad in their range of porosities, gross size, shape and mechanical characteristics, are more difficult to "infiltrate" with aqueous solutions. To increase deposition of solutions into internal surfaces of such materials, hydrophobic materials can be impregnated with the protein, or a surfactant can be used to facilitate impregnation with the protein (e.g. NELL-I). Descriptions of various biodegradable delivery materials comprising materials such as fibrinogen, polylactic acid, porous ceramics, gelatin, agar, and the like can be found, e.g., in U.S. Pat. Nos. 5,736,160; 4,181,983; 4,186,448; 3,902,497; 4,442,655; 4,563,489; 4,596,574; 4,609,551; 4,620,327; and 5,041,138.

Other delivery vehicles include, but are not limited to, bone graft materials. Bone graft materials can be derived from natural materials (e.g., transplanted bone or bone fragments), synthetic materials (e.g., various polymers or ceramics), or combinations of both. Bone graft materials can be used to fill voids or otherwise replace lost bone material. Such graft materials can also be provided as components of prosthetic devices (e.g., bone replacements or supports) to facilitate tight bonding/annealing of the prosthetic with the living bone.

Bone grafts using bioactive glasses, calcium phosphates, collagen, mixtures thereof and the like have good biocompatibility and give rise to bone tissue formation and incorporation in some cases. A number of different glasses, glass-ceramics, and crystalline phase materials have been used, either alone or in combination with acrylic polymerizable species, and other families of polymers for restorative purposes. These include hydroxyapatite, fluorapatite, oxyapatite, Wollastonite, anorthite, calcium fluoride, agrellite, devitrite, canasite, phlogopite, monetite, brushite, octocalcium phosphate, Whitlockite, tetracalcium phosphate, cordierite, and Berlinite. Representative patents describing such uses include U.S. Pat. Nos. 3,981,736; 4,652,534; 4,643,982; 4,775,646; 5,236,458; 2,920,971; 5,336,642; and 2,920,971. Additional references include Japanese Pat. 87-010939 and German Pat. OS 2,208,236. Other references are found in W. F. Brown, "Solubilities of Phosphate & Other Sparingly Soluble Compounds," Environmental Phosphorous Handbook, Ch. 10 (1973). In addition to the foregoing, certain animal-derived materials, including coral and nacre, can also be used in biomaterials for restorative purposes. Other bone graft materials include a pliable, moldable acrylic -based bone cement reinforced with from 15% to 75% by weight of a bioactive glass together with between 1% and 10% by weight of vitreous mineral fibers (U.S. Pat. 4,239,113), bone fillers such as tricalcium phosphate and bioceramic A₂ into bisphenol-A-diglycidyl methacrylate (bis GMA) polymerizable through the action of peroxide systems such as benzoyl peroxide mixed with amines, (Vuillemin et ah, Arch. Otolygol. Head Neck Surg., 113: 836-840 (1987)). Resin composites containing both salicylates and acrylates, cured through a calcium hydroxide cement reaction, are described in U.S. Pat. 4,886,843, while U.S. Pat. Nos. 5,145,520 and 5,238,491 discloses fillers and cements. The foregoing materials can be fabricated so as to incorporate NELL- 1 proteins.

In addition, graft materials that include bone morphogenic proteins are known. For example, U.S. Pat. 4,394,370 describes complexes of reconstituted collagen and demineralized bone particles or reconstituted collagen and a solubilized bone morphogenetic protein fabricated in a sponge suitable for in vivo implantation in osseous defects. U.S. Pat. 5,824,084 describes substrates made from a biocompatible, implantable graft material, preferably having a charged surface. Examples of biocompatible, implantable graft materials include synthetic ceramics comprising calcium phosphate, some polymers, demineralized bone matrix, or mineralized bone matrix. These materials may additionally contain cell adhesion molecules bound to the surface of the substrate. The term "cell adhesion molecules" refers collectively to laminins, fibronectin, vitronectin, vascular cell adhesion molecules (V- CAM) and intercellular adhesion molecules (I-CAM) and collagen. Suitable graft materials include, but are not limited to, isolated mineralized cancerous bone sections, powders or granules of mineralized bone, demineralized cancellous bone sections, powders or granules of demineralized bone, guanidine-HCl extracted demineralized bone matrix, sintered cortical or cancellous bone, coralline hydroxyapatite sold by Interpore under the trade name Interpore 500, and granular ceramics such as that incorporated into the bone graft substitute Collagraft sold by Zimmer, and filamentous sponges such as those made from collagen by Orquest. NELL-I proteins can be incorporated into any of these graft materials or substituted in place of bone morphogenic proteins.

Methods of Use

The composition described herein can be used to treat, prevent, or ameliorate any medical conditions related to angiogenesis by administering to a mammalian subject a composition described above. In some embodiments, a medical condition can be a treated, prevented or ameliorated by enhancing angiogenesis in the subject. Such medical conditions that benefit from stimulation of angiogenesis includes: coronary artery disease (CAD), cardiac failure, tissue injury, chronic wounds, etc. In some embodiments, a medical condition can be a treated, prevented or ameliorated by decreasing or inhibiting angiogenesis in the subject. Such medical conditions that benefit from inhibition of angiogenesis includes: cancer, diabetic nephropathy, arthritis, psoriasis, and possibly various forms of arthritis.

The composition described herein can be delivered or administered to a mammalian subject in any established method of delivery, which can be systemic or local delivery. Systemic delivery includes delivery by e.g., oral administration, sytemic injection, topical delivery or inhalation. Local delivery includes, e.g., implant or local injection. Formulations suitable for systemic or local deliveries are described above.

Kits In other embodiments, the present invention provides kits for practice of the assays or use of the compositions described herein. In one embodiment, the kits comprise one or more containers containing antibodies and/or nucleic acid probes and/or substrates suitable for detection of NELL-I expression and/or activity levels. The kits may optionally include any reagents and/or apparatus to facilitate practice of the assays described herein. Such reagents include, but are not limited to, buffers, labels, labeled antibodies, labeled nucleic acids, filter sets for visualization of fluorescent labels, blotting membranes, and the like.

In another embodiment, the kits comprise a container containing a NELL-I protein, or a vector encoding a NELL-I protein and/or a cell comprising a vector encoding a NELL-I protein. In addition, the kits can include instructional materials containing directions (i.e., protocols) for the practice of the assay methods of this invention or the administration of the compositions described herein along with counterindications. While the instructional materials typically comprise written or printed materials, they are not limited to such. Any media capable of storing such instructions and communicating them to an end user are contemplated by this invention. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD-ROM), and the like. Such media can include addresses to internet sites that provide such instructional materials.

The following example illustrates, but not to limit, the claimed invention. Example 1. The study of NeIl-I and BMP-2 gene modified goat auricular chondrocytes in vivo Material and methods Culture of Primary goat auricular cartilage cells

Adult goat auricular cartilage, isolated without perichondrium, was minced into small pieces and digested with 0.25% trypsin/lmM EDTA at room temperature for 30 min, followed by 3mg/ml collagenase digestion with shaking at 37°C for 6 h. The cell suspension was filtered through a 75um mesh filter and the chondrocytes were then pelleted by centrifugation. After washing with PBS, the cells were cultured in DMEM (Gibco BRL, Grand Island, NY, USA) plus 10% fetal calf serum (Hyclone, Logan, UT, USA), 100 U/ml penicillin and 100 mg/1 streptomycin at 37°C with 5% CO₂. Second passage cells were used for gene transfer studies. Gene transduction of primary cultured chondrocytes

Adenoviral vectors encoding LacZ (AdLacZ), rat NeIl-I (AdNeIl-I), or human BMP- 2 (AdB MP-2) were generated as described previously (Zhang, X., S. Kuroda, et al. (2002). "Craniosynostosis in transgenic mice overexpressing Nell- 1. " J Clin Invest 110(6): 861-70). Chondrocytes were cultured for 24 h to reach 80% confluence and transduced with a multiplicity of infection (MOI) of 50 pfu/cell. Gene transfer efficiency was determined microscopically (Leica DM IRB, Germany) with X-gal staining three days after transduction with AdLacZ by calculating the number of blue stained cells among all the cells observed (Zhang, X., D. Carpenter, et al. (2003). "Overexpression of NeIl-I, a craniosynostosis- associated gene, induces apoptosis in osteoblasts during craniofacial development." J Bone Miner Res 18(12): 2126-34). 50 pfu/cell AdNeIl-I and AdLacZ were chosen for in vitro and in vivo experiments based on transduction efficiency and level of NeIl-I protein production.

To determine the expression of NeIl-I protein, whole cell extracts were prepared from transduced chondrocytes at 72 hours post-transduction. After washing with ice-cold PBS, the cells were lysed using a protein extraction regent (Kangchen Bio-tech, Shanghai, China). Proteins were fractionated by electrophoresis on 6% polyacrylamide gels and transferred to PVDF membranes (Amersham Biosciences, NJ). Membranes were exposed to anti-Nell- 1 (1:850 dilution) and anti-β-actin antibodies (1: 10000 dilution, Sigma, St. Louis, MO). Blots were exposed to secondary goat anti-rabbit for Nell- 1 and anti-mouse for β-actin IgG antiserum conjugated to horse radish peroxidase, and developed with ECL plus chemiluminescence reagent (Amersham Biosciences, Piscataway, NJ). Animal experiments

Chondrocytes three days after gene transduction were used for in vivo experiments. Twelve 5-6 week old BALB/c Nude mice were obtained (Charles River Laboratories, MA). Eight million AdNeIl-I, AdLacZ or AdBMP -2 transduced chondrocytes combined with 160 μl 20% F 127 pluronic solution were injected into nude mice subcutaneous Iy respectively under general anesthesia. For each group, AdNeIl- 1 and AdLacZ transduced cells were injected in one mouse and AdBMP -2 transduced cells were injected in another mouse so as to avoid of any potential synergistic function of NeIl-I with BMP-2 (number of injection sites: AdNell-l=6; AdLacZ=6; AdBMP -2=6). Three mice from each group were sacrificed after two weeks, and the remaining three from each group were sacrificed after four weeks. An extra 2 mice were injected with AdLacZ transduced chondrocytes for x-gal staining after 4 weeks to trace the origin of the injected cells. Each sample from week 4 was weighted and analyzed by ANOVA.

Micro CT and histological analysis of ectopic cartilage formation

The samples were fixed in 10% formalin and those harvested at week 4 were then scanned using microCT to evaluate the mineralization, which utilizes 9-20 μm resolution technology from μCT40 (Scanco Medical, Basserdorf, Switzerland) as previously published (Zhang, 2002). Visualization and reconstruction of the data was performed using the μCT Ray T3.3 and μCT Evaluation Program V5.0 provided by Scanco Medical. After reconstruction of the imagine, the mineralized components for those samples were displayed in red. Bone volume and density from different groups were compared among the groups using single factor ANOVA with the SNK method. For histological analysis, paraffin embedded decalcified samples were sectioned at 5 μm and stained with hematoxylin and eosin. Alcian blue staining was performed to visualize cartilage formation at weeks 2 and 4. X-gal staining was performed on cryosections to determine the presence of LacZ transduced chondrocytes in vivo. Immunohistochemical analysis Paraffin embedded sections were deparaffinized and incubated with primary antibodies including anti-Nell- 1 (1 :850 dilution) (Zhang, 2002), anti-BMP-2 (1:200 dilution), anti-OCN (1 : 100), anti-Cbfal (1: 100), anti-VEGF (1 : 100), and anti-Tenascin-X (1: 100). anti- P-JNK(1 : 100), anti-p-P38(l:50) (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-ColX (1:80 dilution) (Research Diagnostics, Inc., Flanders, NJ). anti-p -MAPK(1: 10O)(CeIl Signaling Technology, Danvers, MA). ABC complex (Vector Laboratories, Burlingame, CA) was applied to the sections following the incubation with biotinylated secondary antibody (Dako Corporation, Carpinteria, CA). AEC plus substrate in red color (Dako) was used as a chromagen, and the sections were counterstained with light Hematoxylin. PBS substituted for the primary antibody was utilized as a negative control.

Results

Culture of Primary goat auricular chondrocytes An MOI of 50 pfu/cell produced optimal effects in transfer efficiency without excessive cell death in vitro. Three days after transduction with 50 pfu/cell AdLacZ, X-gal staining showed that over 80% (looks like 50% in photo) of chondrocytes had successful transduction (Fig. IA). To confirm the production of NeIl-I protein in AdNeIl-I transduced chondrocytes, Western blot analysis was performed. The results revealed increased Nell- 1 production upon transduction with 50 pfu/cell AdNeIl-I, while NeIl-I production in AdLacZ and AdBMP -2 transduced cells remained undetectable (Fig. IB).

NeIl-I induced non-mineralized tissue formation in vivo

Four weeks after injection, AdNeIl-I treated samples had an average weight of

23.6mg, which was 3 times higher than AdLacZ treated samples (7.4 mg) (PO.05), but less than AdB MP -2 samples that had the highest weight at 109 mg (Fig. 2). Further quantitative analysis on bone volume measured from the microCT revealed that the AdB MP -2 group had a significantly larger average volume of 91.1 ±45.9 mm³, with the AdNeIl-I group second with 14.5±8.3 mm , and the AdLacZ group with much less at only 3.4±2.4 mm (Fig. 3B).

MicroCT reconstructions showed that the AdBMP-2 group had large mineralized areas (Fig. 3A) with a significantly larger density of 136.5±37.5 mgHA/mm³. While the other two groups did not show obvious mineralization with barely detectable density values for AdNeIl- 1 group at 6.6±2.2 mgHA/mm and for AdLacZ group at 1.6±2.4 mgHA/mm (Fig. 3C).

NeIl-I and BMP-2 induce hypertrophic cartilage formation at week 2 in vivo

Histological analysis at week 2, chondrocyte condensations formed within AdNeIl-I and AdBMP-2 transduced chondrocyte injection sites and were encapsulated by fibroblastic like cells (Fig. 4A,B), while AdLacZ treated sites showed mainly fibroblastic like tissue (Fig. 4C). Alcian blue staining demonstrated strong GAG production within chondrocytes condensations in AdNeIl-I or AdBMP-2 group, while less GAG production in fibroblastic tissue encapsulating those chondrocyte condensations or in the AdLacZ group (Fig. 4D,E,F). NeIl-I induce cartilage while BMP-2 induced endochondral bone formation at week 4 in vivo At week 4, samples in AdNeIl-I group showed large areas of typical mature cartilage by HE and alcian blue staining (Fig. 5A,D). In the AdBMP-2 group, new bone was formed with blood vessel invasion (Fig. 5B,E), although small area of cartilage remained as demonstrated by alcian blue staining (Fig. 5E). In contrast, AdLacZ samples showed mainly less mature chondrocytes (Fig. 5C). Although alcian blue staining in this group (Fig. 5F) was more pronounced stained than its week 2 samples, it was still much less stronger compared with those in typical mature cartilage areas in AdNeIl-I group at the same time. In vivo NeIl-I and BMP-2 production corresponds with transduction In order to determine the origin of the newly formed cartilaginous and bony tissue in

AdNeIl-I, AdBMP-2, and AdLacZ transduced chondrocytes injection sites after 4 weeks, immunohistochemical analysis for NeIl-I (Fig. 5 G₅H₅I), BMP-2 (Fig. 5 J₅K₅L), and β- galactosidase staining for LacZ (Fig. 5M) were performed. Large numbers of chondrocytes stained positively for NeIl-I in the AdNeIl-I group (Fig. 5G), but not in the AdBMP-2 and AdLacZ groups (Fig. 5 H,I). BMP-2 immunohistochemistry displayed very intense positive staining for samples injected with AdBMP-2 transduced chondrocytes (Fig. 5K), compared to much less obviously detected AdNeIl-I or AdLacZ transduced cells (Fig. 5 J,L). In addition, we also verified by blue X-gal staining that AdLacZ transduced chondrocytes were present intramuscularly and maintained the expression of LacZ four weeks after injection (Fig. 5M).

NeIl-I and BMP-2 stimulate mature cartilage production in vivo

The expression pattern of CoIX, a later marker of chondrogenesis, was evaluated in the cartilaginous and bony tissue with immunohistochemistry. At week 2, the expression of CoIX was pronounced in hypertrophic chondrocytes in both AdNeIl- 1 and AdBMP-2 groups (Fig. 4G,H), but not obviously observed in AdLacZ group (Fig. 41). At week 4, CoIX was intensely stained in hypertrophic chondrocytes in AdBMP-2 and AdNeIl-I groups and in the extracellular matrix of the AdBMP-2 group (Figs. 6D,E). The staining was less expressed in the chondrocytes of the AdLacZ group (Figs.6F)., The expression patterns of Tenascin X, another chondrogenic marker, was pronounced in hypertrophic chondrocytes in AdNeIl-I (Fig. 6G), but only weakly detected in the extracellular matrix of the AdBMP-2 group (Figs.6H,I). NeIl-I signals

Since AdNeIl-I transduction of chondrocytes led to mature hypertropic cartilage and AdBMP-2 transduction of chondrocytes led to mixture of small area of cartilage and bone formation, while the AdLacZ transduced chondrocytes led to fibrous tissue or cartilaginous formation, we attempted to determine the nature of their cartilage or bone formation mediated through a different pathway in the AdNeIl-I and AdBMP-2 groups. To explore the possible mechanism of pathway behind, p-P38,p-MAPK and p-JNK were stained for week 2 smaples. p-JNK was not obviously detected in any groups(data not shown). p-P38 was more pronounced in the cells nuclears in AdBMP-2 group(Figs.4K) than in AdNeIl-I or AdLacZ group(Figs.4J,L). While p-MAPK was a little bit more strongly stained in the chondrocytes nuclears in AdNeIl-I group(Figs.4M) than other two groups(Figs. 4N,O). the expression pattern of Cbfal, a transcription factor that plays a very important role in both osteogenesis and chondrogenesis, was evaluated in the cartilaginous tissue with immunohistochemistry at week 4. Cbfal was detected in the cell nucleus and was more pronounced in AdBMP-2 group (Figs. 6B), but detected to a lesser extent in AdNeIl-I or AdLacZ group (Figs. 6A,C). NeIl-I did not stimulate bone formation by chondroblasts in vivo

AdBMP-2 transduction promoted endochondral ossification as detected by MicroCT. Thus, VEGF and OCN immunohistochemistry was conducted to confirm. As expected, AdBMP-2 samples had strong expression of VEGF which was not detectable in the other groups (Figure 7A,B,C). The osteogenic marker OCN was also strongly stained only in the extracellular matrix from AdBMP-2 group, but not AdNeIl-I or AdLacZ (Figure 7D,E,F). Discussion

This study was to investigate the effect of NeIl-I for promoting cartilage regeneration through regional ex vivo gene therapy with auricular goat chondrocytes. The data showed that the chondrogenic but not osteogenic activity of goat chondrocytes was significantly enhanced by adenoviral gene transfer of NeIl-I, indicating that NeIl-I could be a specific chondrogenesis differentiation molecule in chondrocytes and a candidate for ex vivo gene therapy in regenerating new cartilage.

NeIl-I 's chondrogenic stimulation on goat auricular chondrocytes was tested here, since the goat model could serve as a higher mammal for future studies to regenerate large cartilage (Louwerse, R. T., I. C. Heyligers, et al. (2000). "Use of recombinant human osteogenic protein- 1 for the repair of subchondral defects in articular cartilage in goats." J Biomed Mater Res 49(4): 506-16). In the current study, goat chondrocytes were successfully transduced at a relatively high efficiency of approximately 80% for AdLacZ, AdNeIl-I, and AdBMP-2 at an MOI of 50 pfu/cell. Gene transduction led to the production of NeIl-I or BMP -2 protein in goat auricular chondrocytes in vitro on day 3. In addition, the transduced chondrocytes survived at least four weeks after in vivo injection, verified by either X-gal staining or specific NeIl-I or BMP -2 immunohistochemistry.

Pluronic F 127 was chosen as a carrier because of its previous success in studies to construct tissue engineered cartilage (Liu, Y., F. Chen, et al. (2002). "Repairing large porcine full-thickness defects of articular cartilage using autologous chondrocyte-engineered cartilage." Tissue Eng 8(4): 709-21) and low reactivity in mammals (Cao, Y., A. Rodriguez, et al. (1998). "Comparative study of the use of poly(glycolic acid), calcium alginate and pluronics in the engineering of autologous porcine cartilage." J Biomater Sci Polym Ed 9(5): 475-87). Pluronic F127 consists by weight of approximately 70% ethylene oxide and 30% propylene oxide, making a hydrogel that is slowly dissolved and cleared by renal and biliary excretion (Saim, A. B., Y. Cao, et al. (2000). "Engineering autogenous cartilage in the shape of a helix using an injectable hydrogel scaffold." Laryngoscope 110(10 Pt 1): 1694-7).

The study in this example clearly demonstrates that NeIl-I gene transduction had increased the volume and weight of cartilage formed as compared with the LacZ group. NeIl- 1 promoted chondrocyte differentiation as suggested by CoIX expression, a later marker of chondrogenesis (Pacifici, M., E. B. Golden, et al. (1990). "Hypertrophic chondrocytes. The terminal stage of differentiation in the chondrogenic cell lineage?" Ann N Y Acad Sci 599: 45-57). BMP -2 gene transduction also greatly increased the overall volume and weight of in vivo samples, and it greatly promoted the hypertrophy of chondrocytes by CoIX expression. BMP protein was previously documented to increase the expression of the specific hypertrophic chondrocyte marker type X collagen by inducing type X collagen promoter activity (Shukunami, C, Y. Ohta, et al. (1998). "Sequential progression of the differentiation program by bone morphogenetic protein-2 in chondrogenic cell line ATDC5." Exp Cell Res 241(1): 1-11; VoIk, S. W., P. Luvalle, et al. (1998). "A BMP responsive transcriptional region in the chicken type X collagen gene." J Bone Miner Res 13(10): 1521-9). Because endochondral bone formation is preceded by a chondrogenic phase, it logical that the effect of BMPs involve a chondrogenic response (Hunziker, E. B. (2001). "Growth-factor-induced healing of partial-thickness defects in adult articular cartilage." Osteoarthritis Cartilage 9(1): 22-32) although the cartilaginous tissue was not maintained in these studies. Micro CT reconstruction revealed mineralized components in BMP -2 samples. Vascular epithelial VEGF and the ECM protein osteocalcin are known to play functional roles in cell-matrix interactions during endochondral ossification (Hunziker, 2001). NeIl-I treated samples did not have an obvious VEGF or OC expression, while BMP -2 samples produced both markers. VEGF expression was much more obvious in AdBMP -2 samples indicating BMP-2 would have promoted endochondral osteogenesis through angiogenesis. Angiogenesis, involving the invasion of perichondrium and hypertrophic zone by blood vessels, is required for the replacement of cartilage by bone (Colnot, C, L. de Ia Fuente, et al. (2005). "Indian hedgehog synchronizes skeletal angiogenesis and perichondrial maturation with cartilage development." Development 132(5): 1057-67). The angiogenic factor, VEGF, promotes vascular invasion via specifically localized receptors, including FIk that is expressed in endothelial cells in the perichondrium or surrounding soft tissues and neuropilin (Npn) 1 that is expressed in late hypertrophic chondrocytes (Colnot, C. I. and J. A. Helms (2001). "A molecular analysis of matrix remodeling and angiogenesis during long bone development." Mech Dev 100(2): 245- 50). There are controversial data concerning the effect of BMP-2 on the terminal differentiation of chondrocytes indicating that a variety of co-factors may be involved in this process. BMP induced bone formation may particularly depend on vascularization, because the simultaneous application of anti angiogenic agents in animal studies could suppress osteogenesis and preserve the differentiation state of chondrocytes. (Hunziker, 2001; Takita, H., M. Kikuchi, et al. (2002). "Inhibition of BMP-induced ectopic bone formation by an antiangiogenic agent (epigallocatechin 3-gallate)." Connect Tissue Res 43(2-3): 520-3). That this process did not proceed to bone formation in a certain partial thickness articular cartilage defect model is most likely attribute to the absence of blood vessels within such a lesion type. Osteogenic precursor cells derived from the perivascular spaces are essential for bone formation (Hunziker, 2001). That fact that NeIl-I promoted chondrogenesis, but not osteogenesis, in goat auricular chondrocytes may be due to the lack of VEGF induction leading to no detectable angiogenesis even with exposer to the vascular system.

Tenascin was used as a chondrogenic differentiation marker (Iwamoto, M., E. Koyama, et al. (2005). "The balancing act of transcription factors C- 1-1 and Runx2 in articular cartilage development." Biochem Biophys Res Commun 328(3): 777-82). In this study, this marker was only detectable in NeIl-I treated chondrocytes indicating that those cells maintained a cartilaginous phenotype. This finding is extremely interesting since the data coincided with another report in a NeIl-I deficiency animal (Nell- 16R) model. In that study, the loss of NeIl-I function lead to skeletal defects in the cranial vault, vertebral column and ribcage. Gene expression assays revealed that these aberrant phenotypes were because of the down regulation of extracellular matrix, cell adhesion, and cell communication proteins necessary for osteogenesis and chondrogenensis. Interestingly, expression levels of tenascin were affected in both head and bodies in Nell- 16R mice. Those data suggested that NeIl-I had a critical function in normal chondrogenesis in addition to its role in intramembranous and endochondral bone formation (Desai, J., M. E. Shannon, et al. (2006). "NeM -deficient mice have reduced expression of extracellular matrix proteins causing cranial and vertebral defects." Hum MoI Genet 15(8): 1329-41).

It has been firmly established that Smad pathways are central mediaors of signals from the receptors for BMPs, however, growing biochemical and developmental evidence supports the notion that alternative, non-Smad pathways also participated in BMPs signaling (Moustakas, A. and C. H. Heldin (2005). "Non-Smad TGF -beta signals." J Cell Sci 118(Pt 16): 3573-84). In our previous study, NeIl-I did not show its involvement of Smad signalling for C2C12 myblasts(data not shown). As a preliminary exploration of the possible mechanism behind the difference of NeIl-I with BMP -2 in chondrogenesis or endochondral osteogenesis, three main MAPK signal pathway markers p-P38, p-JNK and p-ERKl/2 immunohistochemistry staining were performed at week 2 after implantation. In all three groups, p-JNK was negative. It coincides with the description that JNK phosphorylation is not affected during chondrogenesis, suggesting that JNKs plays only minor roles in this process (Nakamura, H., A. Kawakami, et al. (1999). "Expression of mitogen activated protein kinases in labial salivary glands of patients with Sjogren's syndrome." Ann Rheum Pis 58(6): 382-5). In contrast, p38 and ERKs occupy central positions in this process (Stanton, L. A., T. M. Underhill, et al. (2003). "MAP kinases in chondrocyte differentiation." Dev Biol 263(2): 165-75). Reilly demonstrate that ERKl/2 plays a negative role while p38 plays a positive role in the BMP -2 activated transcription of type X collagen (Reilly, G. C, E. B. Golden, et al. (2005). "Differential effects of ERK and p38 signaling in BMP-2 stimulated hypertrophy of cultured chick sternal chondrocytes." Cell Commun Signal 3(1): 3). We found p-P38 was more pronounced in BMP-2 group while p-ERKl/2 was a little more pronounced in NeIl-I group. It suggested those two factors might have gone through different pathway in non smad MAPK pathway during chondrogenesis.

Cbfal detection at week 4 revealed an appreciable upregulation in the BMP-2 group than in the NeIl-I group. This is consistent with the report that NeIl-I is an immediate downstream mediator of Cbfal/Runx2 (Truong, T., X. Zhang, et al. (2007). "Craniosynostosis-associated gene nell-1 is regulated by runx2." J Bone Miner Res 22(1): 7- 18). However, the real mechanism of Nell-1 signaling is still largely unknown and needs further exploration in future studies. Conclusions Nell-1 promoted chondrogenic but not osteogenic differentiation of goat auricular chondrocytes. This differed from BMP -2, which lead to endochondral ossificationand might have functioned through a different signaling pathway. The data indicates that Nell-1 is effective promote cartilage regeneration through an ex vivo regional gene therapy method. Example 2. Decreased Proliferation in Human Tumor Cell Lines Saos-2 (American Type Culture Collection) human osteosarcoma cells were cultured in DMEM with 10% FBS, 100 units/ml penicillin, and 100 g/ml streptomycin. Recombinant human Nell-1 protein was added in ranges of 100 ng/ml to 1.6 microgram/ml. Maximal inhibition of Saos-2 cell proliferation was noted on day 8 using cell count or commercial cell proliferation assays. It is understood that the example and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be apparent to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Claims

WE CLAIM:

1. A method of inhibiting angiogenesis in a mammalian subject, comprising administering to the subject an antiangiogenic composition comprising a NELL-I protein, wherein the NELL-I protein is in an amount effective for inhibiting vascular formation in a tissue in the subject.

2. The method of claim 1, wherein the NELL-I protein includes a modulated functional domain such that the antiangiogenic effect of the NELL-I is augmented.

3. The method of claim 2, wherein the modulated is a TSP-I like domain.

4. The method of claim 1 or 2, wherein the composition is effective for cartilage formation.

5. The method of claim 1 or 2, wherein the composition is effective for antineoplastic application.

6. The method of claim 1 or 2, wherein the composition is effective for anti-tumor application.

7. A method of promoting angiogenesis in a mammalian subject, comprising administering to the subject an angiogenic composition comprising a NELL-I protein, wherein the NELL-I protein includes a modulated functional domain such that the angiogenic effect of the NELL- 1 is augmented, and wherein the NELL- 1 protein is in an amount effective for promoting vascular formation in a tissue in the subject.

8. The method of claim 7, wherein the modulated functional domain is a TSP-I like domain such that the antiangiogenic effect of the TSP-I like domain is negated.

9. The method of claim 7 or 8, wherein the composition is effective for enhancing bone formation in the tissue.

10. The method of claims 1 or 7, wherein the mammalian subject is a patient.

11. An antiangiogenic composition, comprising a NELL- 1 protein, wherein the NELL- 1 protein includes a modulated functional domain such that the antiangiogenic effect of the NELL-I is augmented.

12. The antiangiogenic composition of claim 11 wherein the composition is effective for cartilage formation.

13. The antiangiogenic composition of claim 11, wherein the composition is effective for antineoplastic application.

14. The antiangiogenic composition of claim 13, wherein the composition is effective for anti-tumor application.

15. The antiangiogenic composition of claim 14, wherein the composition is effective for anti-cancer application.

16. An angiogenic composition, comprising a NELL-I protein, wherein the NELL- 1 protein includes a modulated functional domain such that the angiogenic effect of the NELL- 1 is augmented, and wherein the NELL- 1 protein is in an amount effective for promoting vascular formation in a tissue in the subject.

17. The angiogenic composition of claim 16, wherein the modulated functional domain is a TSP-I like domain such that the such that the antiangiogenic effect of the TSP-I like domain is negated.

18. The angiogenic composition of claim 16, wherein the composition is effective for enhancing bone formation in the tissue.