EP1603587A1 - Use of tumor endothelial markers 1, 9 and 17 to promote angiogenesis - Google Patents

Abstract

The methods and compositions provided herein promote angiogenesis using exogenous expression of Tumor Endothelial Marker (TEM) 1, TEM 9, or TEM 17 protein, or biologically active fragments thereof, in endothelial cells. Also provided are methods to identify inhibitors of angiogenesis in mice transgenic for TEM 1, TEM 9, or TEM 17 proteins. Therefore, these methods and compositions are useful in the accelerating or augmenting wound healing, treatment of anoxic tissues (e.g., cardiac muscle), ulcers, and transplanted tissues, particularly vascular grafts, in vertebrates.

Description

USE OF TUMOR ENDOTHELIAL MARKERS 1 , 9 AND 17 TO PROMOTE ANGIOGENESIS

Technical Field

[0001] The methods and compositions provided herein promote angiogenesis and thus are useful in the accelerating or augmenting wound healing, treatment of anoxic tissues (e.g., cardiac muscle), ulcers, and transplanted tissues, particularly vascular grafts, in vertebrates. More specifically, the methods relate to the promotion of angiogenesis by the exogenous expression of a tumor endothelial marker (TEM) in an endothelial cell, and compositions useful in these methods. Also provided are methods to identify inhibitors of angiogenesis in mice transgenic for TEM proteins.

Background

[0002] Angiogenesis encompasses the generation of new blood vessels in a tissue or organ. Under normal physiological conditions, angiogenesis occurs in very specific situations such as wound healing, fetal development, and the formation of the corpus luteum, endometrium and placenta. The process of angiogenesis is highly regulated through a system of naturally occurring stimulators, e.g., angiopoietin-1, IL-8, platelet-derived endothelial cell growth factor (PD-ECGF), and tumor necrosis factor-alpha (TNF- α), and inhibitors, e.g., thrombospondin, interferon, and metalloproteinase inhibitors.

[0003] Angiogenesis also can occur as a significant factor in a number of disease states. In fact, uncontrolled angiogenesis directly contributes to the pathological damage associated with many diseases. This uncontrolled or excessive angiogenesis occurs when an imbalance in the angiogenic factors and angiogenic inhibitors occurs, e.g., when an excessive amount of angiogenic factor is produced. Insufficient angiogenesis also contributes to certain disease states. For example, inadequate blood vessel growth contributes to the pathology associated with coronary artery disease, stroke, and delayed wound healing.

[0004] Normal wound healing is responsible for the rapid restoration of tissue integrity and function following a variety of insults, including trauma, burns, and infection. The induction of angiogenesis is a critical component of the wound healing process. Angiogenesis ensures that proliferating and differentiating endothelial cells, pericytes, and fibroblasts are supplied with nutrients and oxygen, and that immune effectors of humoral and cellular immunity are delivered to the wound to ward off potential infection. Therefore, it is desirable to induce neoangiogenesis as early as possible in the course of wound healing, particularly in the case of patients having conditions that tend to retard wound healing, e.g., burns, decubitis ulcers, diabetes, obesity and malignancies. Similarly, an acceleration of wound healing benefits patients post-surgery as well. [0005] Another disease associated with ischemic tissue damage is coronary artery disease. Coronary artery disease usually results from blockages in the large and medium-sized arteries supplying the heart that in turn cause myocardial ischemia. In myocardial ischemia, a limited and/or irregularly distributed blood flow from the coronary arteries to heart tissue results in tissue anoxia. In severe cases, such anoxia can result in widespread cell death in the tissues, ultimately causing myocardial infarction (heart attack) or sudden cardiac death. Another disease associated with myocardial ischemia is angina pectoris, a chronic condition characterized by chest discomfort as a result of exertion. In these disease states, and other like them, the ability to stimulate angiogenesis can significantly decrease the long term tissue damage by reversing the anoxic state through the restoration of blood flow to the ischemic tissue beds.

[0006] Normally in the adult mammalian organism, angiogenesis occurs infrequently. However, it can be rapidly induced in response to a number of diverse physiologic stimuli as described above. Initially, the endothelial cells and pericytes, surrounded by a basement membrane, form capillary blood vessels. Disease or injury induces the production of pro-angiogenic factors. Initially, these factors activate endothelial cells and leukocytes to release enzymes that erode or dissolve the basement membrane of the existing blood vessels. The activated endothelial cells, which line the lumen of blood vessels, then protrude through the basement membrane. Pro-angiogenic stimulants then induce the endothelial cells to migrate through the compromised basement membrane. The migrating cells form a "sprout" off the parent blood vessel, where the endothelial cells begin to proliferate. The endothelial sprouts merge with each other to form capillary loops using adhesion factors, creating new blood vessels. Additional enzymes, e.g., matrix metalloproteinases, then digest the tissue at the tip of the sprouting vessel, permitting active tissue remodeling around the new vessel. The newly formed vessels are stabilized by the pericytes (i.e., specialized smooth muscle cells). Once stabilized, the new vessels support blood flow.

[0007] A number of growth factors have been identified as useful in stimulating angiogenesis. These include fibroblast growth factor (FGF) family (Yanagisawa-Miwa, et al, Science, 257:1401-1403 (1992) and Baffour, et al, J Vase Surg, 16:181-91 (1992)), endothelial cell growth factor (ECGF)(Pu, et al, J. Surg. Res., 54:575-83 (1993)), and vascular endothelial growth factor (VEGF) (Takeshita, et al, Circulation, 90:228-234 (1994) and Takeshita, et al, JClin. Invest, 93:662-70 (1994). However, because many of these pro-angiogenic factors act on a wide variety of tissues, the identification of specific proteins to enhance angiogenesis offer significant therapeutic advantages.

Summary

[0008] In one aspect, the methods and compositions provided herein stimulate endothelial cell proliferation comprising administering to an endothelial cell an effective amount of a tumor endothelial marker (TEM) 9 protein, or a biologically active protein fragment thereof, whereby the endothelial cell is stimulated to proliferate. In one embodiment the TEM protein is administered as a vector comprising a nucleic acid encoding the TEM protein or a biologically active protein fragment thereof. Typically the vector is a viral vector. In one specific embodiment the embodiment is a replication deficient adenovirus.

[0009] In another aspect, the methods and compositions provided herein stimulate endothelial cell proliferation comprising administering to an endothelial cell an effective amount of a tumor endothelial marker (TEM) 17 protein, or a biologically active protein fragment thereof, whereby the endothelial cell is stimulated to proliferate.

[0010] In yet another aspect, the methods and compositions herein stimulate endothelial cell migration comprising administering to an endothelial cell an effective amount of a tumor endothelial marker (TEM) 9 protein, or a biologically active protein fragment thereof whereby the endothelial cell is stimulated to migrate.

[0011] Further provided herein is a method stimulating endothelial cell tubule formation comprising administering to an endothelial cell an effective amount of a tumor endothelial marker (TEM) 1 protein, or a biologically active protein fragment thereof whereby the endothelial cell is stimulated to form tubules.

[0012] In another aspect, the methods and compositions herein stimulate endothelial cell tubule formation comprising administering to an endothelial cell an effective amount of a tumor endothelial marker (TEM) 17 protein, or a biologically active protein fragment thereof whereby the endothelial cell is stimulated to form tubules. In one specific embodiment, the method provided herein further comprises administering colon carcinoma-conditioned media.

[0013] In one aspect, the methods and compositions provided herein stimulate angiogenesis, comprising administering to a subject in need thereof, an effective amount of a vector containing a DNA sequence expressing (tumor endothelial marker) TEM 1 protein, or a biologically active protein fragment thereof, whereby endothelial cells of the subject are infected by said vector and angiogenesis is stimulated. In one embodiment of the present methods, the infection of endothelial cells with the TEM 1 -expressing vector results in increased proliferation and increased tube formation.

[0014] In another aspect, the methods and compositions provided herein stimulate angiogenesis, comprising administering to a subject in need thereof, an effective amount of a vector containing a DNA sequence expressing TEM 9 protein, or a biologically active protein fragment, whereby endothelial cells of the subject are infected by said vector and angiogenesis is stimulated. In one embodiment, the infection of endothelial cells with TEM 9-expressing vector results in increased proliferation and migration.

[0015] In yet another aspect, the methods and compositions provided herein stimulate angiogenesis, comprising administering to a subject in need thereof, an effective amount of a vector containing a DNA sequence expressing TEM 17 protein, or a biologically active protein fragment, whereby endothelial cells of the subject are infected by said vector and angiogenesis is stimulated. In one embodiment, the infection of endothelial cells with TEM 17-expressing vector results in increased tube formation.

[0016] In one embodiment of the above methods, the vector is a viral vector. In specific embodiment, the vector is a replication deficient adenovirus.

[0017] In one embodiment, the subject has anoxic or ischemic tissue damage. In another embodiment, the subject has chronic wound healing deficiencies.

[0018] In one aspect, provided herein is a recombinant virus construct comprising a DNA sequence encoding tumor endothelial marker (TEM) 1, or a biologically active fragment thereof. In one embodiment, the virus is a replication deficient adenovirus.

[0019] In another aspect, provided herein is a recombinant virus construct comprising a DNA sequence encoding tumor endothelial marker (TEM) 9, or a biologically active fragment thereof. In one embodiment, the virus is a replication deficient adenovirus.

[0020] In yet another aspect, provided herein is a recombinant virus construct comprising a DNA sequence encoding tumor endothelial marker (TEM) 17, or a biologically active fragment thereof. In one embodiment, the virus is a replication deficient adenovirus.

[0021] Thus, the methods and compositions provided herein stimulate angiogenesis where tissues are anoxic or ischemic due to injury, surgery, or disease or where accelerated wound healing is recommended by exogenous expression of TEMs in endothelial cells.

Detailed Description

[0022] The methods and compositions disclosed herein relate to the growth and function of the endothelial cell in vitro and in vivo. Specifically, these methods and compositions employ a subset of surface molecules on endothelial cells that are preferentially expressed on endothelial cells found within tumors and are known as tumor endothelial markers or TEMs. The increased expression of one or more these TEMs promote angiogenesis and thus provide useful therapeutic agents for the treatments of anoxic tissue-related injury and disease as well as for the promotion of wound healing.

[0023] A tumor endothelial marker can be expressed in endothelial cells using the methods and compositions provided herein. As used herein, the term "tumor endothelial marker (TEM)" refers to a molecule preferentially expressed on tumor endothelial cells. TEM expression is absent or significantly lower on normal (non-tumor) vasculature. See, e.g., St. Croix, et al, Science 289: 1197-1202 (2000), U.S. Serial No. 09/918715 (Publication No. 20030017157). While it is contemplated that TEMs share a similar expression profile (i.e., they are largely restricted to tumor endothelial cells), TEMs will not necessarily share the same mechanism in promoting angiogenesis. Therefore, the pro-angiogenic properties of one TEM can be distinct, overlapping, or identical with another TEM. The pro-angiogenic properties include, but are not limited to the stimulation of in vitro proliferation, migration, and tube formation by endothelial cells.

[0024] In one embodiment, the target molecule is TEM 1. Endosialin is a 165 kDa glycoprotein. Rettig, et al, Proc. Nat'lAcad. Sci. U.S.A. 89: 10832-36 (1992). In 2001, Christian and others identified the DNA sequence of TEM 1 reported by St. Croix et al. in Science 289: 1197-1202 (2000) as the protein coding sequence for endosialin. Christian, et al, J. Biol. Chem. 276: 7408-14 (2001). TEM 1 is a C-type lectin-like, type I membrane protein with a signal leader peptide, five globular extracellular domains, followed by a mucin-like region, a transmembrane segment and a short cytoplasmic tail. The N-terminal shows homology to thrombomdulin, a receptor involved in regulating blood coagulation and to complement receptor ClqRp. Webster, et al, J. Leuk. Biol. 67: 109-16 (2000). Murine and human TEM 1 share 77.5% amino acid identity with 100% identity in the transmembrane region. Opavsky, et al, J. Biol. Chem. 276: 38795-807 (2001). TEM 1 has a signal sequence at amino acids 1-17 and its transmembrane domain at amino acids 686-708. Its extracellular domain is at residues 1-685. TEM 1 expression varies with cell density (or cell cycle). Opavsky et al, supra (2001). TEM 1 is maximally expressed in confluent (Go) cells, the most relevant phase of the cell cycle in vivo. The DNA sequence of TEM 1 is disclosed as SEQ ID NO. 196 in U.S. Serial No. 09/918715 (Publication No. 20030017157).

[0025] In another embodiment, the target molecule is TEM-17. TEM 17 was a Type I membrane protein with a signal sequence at residues 1-18, a N-terrninal region similar to the Gl domain of nidogen, a 100 amino acid region with homology to plexins, a transmembrane domain at residues 427-445, and a short cytoplasmic tail. Its extracellular region comprises residues 1-426. The plexin family mediates cell guidance cues, suggesting that TEM 17 may function in this capacity. Murine TEM 17 has 81% sequence identity with the human TEM 17. The DNA sequence of TEM 17 is disclosed as SEQ ID NO. 230 in U.S. Serial No. 09/918715 (Publication No. 20030017157).

[0026] In another embodiment, the target molecule is TEM 9. TEM 9 is a secretin family seven- span transmembrane G-protein coupled receptor. This G- protein coupled receptor homologue has both a signal sequence at residues 1-26 and 7 transmembrane domains. The N-terminal extracellular domain consists of a leucine-rich repeat (LRR) region, followed by an immunoglobulin domain, a peptide hormone receptor domain, and a GPCR proteolytic site domain. Its extracellular region resides in amino acids 1-769, and its transmembrane domains are at residues 817-829 (TM2 and TM3), residues 899-929 (TM4 and TM5), and residues 1034-1040 (TM6 and TM7). The transmembrane region is homologous to the transmembrane region of other secretin-family serpentine receptor. The beginning of the cytoplasmic region contains two vicinal cysteines typical for the GPCR-palmitoylation. Thus, TEM 9 is a G-protein coupled receptor with extracellular domains characteristic of cell adhesion proteins. The mouse ortholog has a predicted signal peptide at residues 1-29 and has 87% homology to the human TEM 9. The DNA sequence of TEM 9 is disclosed as SEQ ID NO. 212 in U.S. Serial No. 09/918715 (Publication No. 20030017157).

[0027] Any biologically active fragment of a TEM can be used in the present methods and compositions. As used herein, the term "biologically active fragment" refers to any portion of the TEM protein, and its corresponding encoding DNA sequence, that retains one or more of the biological activities of the full-length protein. Such fragments can include only a part of the full-length sequence and yet possess the same function, possibly to a greater or lesser extent. Such fragments can be evaluated for biological activities using the methods provided herein to assess angiogenesis. Biological activities associated with pro-angiogenic activity include proliferation, migration, and tube formation by endothelial cells in vitro.

[0028] Any analog or derivative of the TEM protein can be used in the methods herein. As used herein, the term "analog or derivative" refers to substituted proteins characterized by the ability to promote angiogenesis as indicated. Such mutations and substitutions can be designed and expressed by well-known laboratory methods and include conservative mutations and substitutions known to the skilled artisan. For example, deletion mutants of a TEM can be designed and expressed by well known laboratory methods. Such analogs and derivatives can be evaluated for angiogenic properties routinely using the assays provided herein as an indicator of biological activity.

[0029] The TEM protein or polypeptide can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Typically, high performance liquid chromatography (HPLC) is employed for purification. Polypeptides useful in the methods provided herein include: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, but not limited to bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides useful in the present methods may be glycosylated or may be non-glycosylated. In addition, TEM polypeptides may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N- terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N- terminal methionine is covalently linked. [0030] It also will be recognized by one of ordinary skill in the art that some amino acid sequences of the TEM polypeptide can be varied without significant effect of the structure or function of the protein. Typically, conservative substitutions include the replacement of, one for another, among the aliphatic amino acids Ala, Val, Leu and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.

[0031] To improve or alter the characteristics of TEM polypeptides, protein engineering may be employed. Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or muteins including single or multiple amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides can show, e.g., enhanced activity or increased stability. In addition, they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.

[0032] The DNA sequences useful in the present methods and compositions include any sequence that encodes a biologically active full length TEM, fragment, analog, or derivative thereof. The sequence may comprise a genomic sequence or a non-genomic sequence. Typically, the sequences will be a cDNA sequence. Exemplary sequences are found in U.S. Serial No. 09/918715 (Publication No. 20030017157).

[0033] Exogenous expression of the TEM protein can be transient, stable, or some combination thereof. Exogenous expression can be enhanced or maximized by co-expression with one or more additional proteins that increase its angiogenic activity.

[0034] In the methods of stimulating angiogenesis, proliferation, migration, and/or endothelial tubule formation, any cell that expresses the TEM of interest, can be induced to express the TEM of interest, or expresses a non-human TEM homologue (e.g., murine, bovine, and the like) may be used. In one embodiment, the cell is a cell line that endogenously expresses the TEM of interest. In a specific embodiment, the cell line is the murine 2H11 endothelial cell line (ATCC). In another embodiment, the cell is induced to express the TEM of interest. In a specific embodiment, the cell is an AC133+/CD34+ human bone marrow cell cultured in the presence of basic FGF (bFGF), VEGF, and heparin to generate the endothelial precursor cell (EPC's). In yet another embodiment, the cell is a transfected or transduced with the TEM of interest. In a specific embodiment, the human umbilical vein endothelial cell (HUVEC) or the human microvascular endothelial cell is transduced with an adenoviral vector encoding the TEM of interest. In another specific embodiment, the COS or 293 cell is transfected with a plasmid encoding the TEM of interest. In some embodiments, the TEM-expressing cell may be contacted with growth factors or media conditioned by other cells. For example, colon carcinoma conditioned media can be prepared using confluent cultures of human HCT116 colon carcinoma cells or human HT29 colon carcinoma cells grown in serum free media for 3 days. Factors useful in supplementing media include VEGF and bFGF. [0035] In one embodiment, the cell is derived from or is in a human or veterinary subject. The endothelial cells can be in a tissue or a subject. The endothelial cells targeted by the methods provided herein can be precursors that can be induced to or will differentiate into functional endothelial cells. The endothelial cells can also be mature endothelial cells. The identification of endothelial cells can be performed using conventional methods, e.g., staining by antibodies specific for endothelial markers.

[0036] The endothelial cell can be contacted with the TEM in any suitable manner for any suitable length of time. Typically, the time of contact with the TEM protein or DNA sequence is sufficient to elicit a pro-angiogenic response. Such a response includes increased proliferation, migration, and/or tube formation by the TEM-expressing endothelial cell.

[0037] Provided herein is a method of stimulating proliferation in an endothelial cell, comprising administering an effective amount of a TEM protein, or a biologically active fragment thereof, whereby the endothelial cell is stimulated to proliferate. The TEM protein can be TEM 1 or TEM 9. In one embodiment, the method of stimulating proliferation in an endothelial cell comprises one or more of the steps of contacting the endothelial cell with the TEM protein, or a biologically active fragment thereof, for a certain duration, and then determining the proliferation of the TEM-expressing endothelial cell relative to the non-TEM expressing endothelial cell, wherein a greater amount of proliferation in TEM- expressing cells indicates that exogenous TEM expression is stimulatory for endothelial cell proliferation. A TEM protein is stimulatory for proliferation if it stimulates proliferation relative to the proliferation of endothelial cells receiving no TEM protein, an inactive TEM protein, or an empty delivery vehicle (e.g., empty vector, empty liposome). The cell can be contacted with the TEM protein for any suitable amount of time.

[0038] Proliferation may be quantified using any suitable methods. Typically, the proliferation is determined by assessing the incorporation of radioactive-labeled nucleotides into DNA (e.g., ³H- thymidine). In one embodiment, proliferation is determined by ATP luminescence. In a specific embodiment, proliferation is determined using the CellTiter-Glo™ Luminescent Cell Viability Assay (Promega). The proliferation of an endothelial cell may also be stimulated by the administration of the TEM protein or encoding DNA sequence to a tissue or to a subject in a manner described below.

[0039] Provided herein is a method of stimulating endothelial cell migration, comprising administering to an endothelial cell, an effective amount of a TEM protein, or a biologically active fragment thereof, whereby the endothelial cell is stimulated to migrate. In one embodiment, the TEM protein is TEM 9. In one embodiment, the method of stimulating endothelial cell migration comprises at least of the steps of contacting endothelial cells with a TEM protein or DNA sequence for a time sufficient to induce TEM expression, incubating the TEM-expressing cells in a first chamber separated from a second chamber by a porous membrane, wherein the second chamber comprises media supplemented with at least one chemoattractant, and determining the number of TEM-expressing cells entering the second chamber relative to the number of non-TEM expressing cells entering the second chamber, wherein a greater number of TEM-expressing cells entering the second chamber indicates that exogenous TEM expression stimulates endothelial cell migration. The non-TEM expressing cells can be untreated endothelial cells or endothelial cells treated with the empty vehicle.

[0040] Any suitable method for assessing migration can be employed with the present methods. Typically, migration is determined by assessing the number of cells in the chamber with the chemoattractant versus those remaining in the original chamber. In one embodiment, the cells are pre- labeled with PKH67 green dye, according to manufacturer's instructions. In another embodiment, the cells are labeled with a fluorescent tag, e.g., Calcein AM (Molecular Probes). Following incubation, the cells are enumerated using a fluorescent inverted phase microscope. The migration of a cell may also be stimulated by the administration of the TEM protein or encoding DNA sequence to a tissue or to a subject in a manner described below.

[0041] Also provided herein is a method of stimulating endothelial cell tubule formation, comprising administering to an endothelial cell, an effective amount of a TEM protein, or a biologically active fragment thereof, whereby the endothelial cell is stimulated to form tubules. In one embodiment the TEM is TEM 1. In another embodiment the TEM is TEM 17. In one embodiment, the method of stimulating endothelial cell tubule formation comprises at least one of the steps of contacting endothelial cells with TEM protein or DNA sequence for a time sufficient to induce TEM expression, incubating the endothelial cells in an appropriate matrix, determining tubule formation, wherein the greater tubule formation by TEM-expressing endothelial cells relative to non-TEM expressing cells indicates that exogenous TEM expression is stimulatory for endothelial cell tubule formation. Typically, tubule formation is stimulated when the number of tubules formed increases or the character of the tubules is significantly altered when the TEM is expressed by the endothelial cell. The non-TEM expressing cells can be untreated endothelial cells or endothelial cells treated with the empty vehicle.

[0042] Any suitable method to assess tubule formation is useful with the methods provided herein. Typically, tubule formation is assessed by microscopy using cells labeled with a detectable label. In one embodiment, the cells are pre-labeled with PKH67 green dye, according to manufacturer's instructions. In another embodiment, the cells are labeled with Calcein AM (Molecular Probes). Following incubation, the endothelial cell tubules are examined using a fluorescent inverted phase microscope. As used herein, the term "character of the tubule" refers to the robustness and duration of the tubule networks formed in the matrix. Tubule formation may be quantified using any suitable methods. In one embodiment, the tubule formation is quantified by assessing tube area using the MetaMorph Image Analysis system. Any suitable matrix may be employed. In one embodiment, the matrix is reconstituted basement membrane Matrigel™ matrix (BD Sciences). The tubule formation by a cell also may be stimulated by the administration of the TEM to a tissue or to a subject in the manner described below. [0043] In a specific embodiment, the TEM is TEM 17, and the method of stimulating endothelial tube formation further comprises supplementing the media with colon carcinoma conditioned media. Typically, the colon carcinoma cell lines used to condition the media are HCT116 and HT29.

[0044] Angiogenesis can be promoted using the present methods and compositions in any situation where a need for angiongenesis is indicated. As used herein, the term "angiogenesis" refers to the growth of blood vessels in tissue or in culture in response to stimuli, particularly the exogenous expression of TEMs. As used herein, the term "neoangiogenesis" refers to the new growth of blood vessels in tissue or in culture. In the methods of stimulating angiogenesis, any cell that can expresses the TEM of interest or that can be induced to express the TEM of interest can be used. In one embodiment, the cell is found in a human or veterinary subj ect. i

[0045] As used herein, the term "tissue" refers to any tissue in which angiogenesis may be desired. Tissues to be treated by the present methods and compositions will typically be adjacent to blood vessels, more typically being adjacent to coronary and peripheral arteries, where, for example, the angiogenic factor can delivered transmurally within the adjacent blood vessel to promote angiogenesis from the delivery site within the blood vessel into the surrounding tissue. The target tissue will usually be ischemic, but the present methods are also useful in promoting angiogenesis in non-ischemic tissues.

[0046] Any tissue ischemia can be treated using the present methods and compositions. As used herein, the term "ischemic" or "anoxic" refers to a tissue having a deficiency in blood flow as the result of disease or injury. Such tissues include, but are not limited to muscle, brain, kidney and lung. Ischemic diseases include, but are not limited to cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy and myocardial ischemia.

[0047] Any wound or other condition requiring or benefiting from neovascularization can be treated with the present methods and compositions. As used herein, the term "wound" refers to any opening in the skin, mucosa or epithelial linings, including openings generally being associated with exposed, raw or abraded tissue. There are no limitations as to the type of wound or other traumata that can be treated in accordance with these methods and compositions. Such wounds include, but are not limited to first, second and third degree burns; surgical incisions, including those of cosmetic surgery; wounds, including lacerations, incisions, and penetrations; and ulcers including decubital ulcers (bed-sores) and ulcers or wounds associated with diabetic, dental, haemophilic, malignant and obese patients. Although the primary concern is the healing of major wounds by neovascularization, it is contemplated that TEM expression also may be useful for minor wounds, vascular and skin grafts, and for cosmetic regeneration of epithelial cells. Typically, the wounds to be treated are burns and surgical incisions, whether or not associated with viral infections or tumors.

[0048] As used herein, the terms "stimulating angiogenesis" and "promoting angiogenesis" include increasing the proliferation, migration, tube formation, or some combination thereof of TEM-expressing endothelial cells. In vivo angiogenesis includes any stimulation of endothelial cells that promotes at least some neoangiogenesis or wound healing. Thus, the term denotes a beneficial result conferred on a vertebrate subject with an ischemia-related disease or symptom, or a wound. In one embodiment, where the TEM is TEM 1, the pro-angiogenic activity includes stimulating proliferation and stimulating tube formation. In another embodiment, where the TEM is TEM 9, the pro-angiogenic activity includes stimulating proliferation and stimulating migration. In yet another embodiment, where the TEM is TEM 17, the pro-angiogenic activity includes stimulating tube formation.

[0049] Any subject can be treated with the methods and compositions provided herein. Such a subject is a mammal, preferably a human, with an ischemia-associated disease or symptom, or a wound. In one specific embodiment, the subject has chronic wound healing deficiencies. Veterinary uses of the disclosed methods and compositions are also contemplated. Such uses would include treatment of ischemia-related diseases and wounds, in domestic animals, livestock and thoroughbred horses.

[0050] Biologically effective amounts will be those amounts that promote at least some wound- healing or neoangiogenesis, with amounts that result in significant improvement of the healing process being preferred. As used herein, the term "effective amount" refers to a sufficient amount of compound (e.g., nucleic acid, protein) delivered to produce an adequate level of the TEM protein, i.e., levels capable of inducing endothelial cell growth and/or inducing pro-angiogenic activities. As used herein, the term "therapeutically effective amount" refers to an amount of a TEM protein that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject is effective to enhance or stimulate angiogenesis in a cell, tissue, or subject benefited by angiogenesis. A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

[0051] Thus, the important aspect is the level of TEM expressed. Accordingly, one can use multiple transcripts or one can have the gene under the control of a promoter that will result in high levels of expression. In an alternative embodiment, the gene would be under the control of a factor that results in extremely high levels of expression. The effective dose of the nucleic acid will be a function of the particular expressed protein, the target tissue, the patient and his or her clinical condition. Typically, effective amounts of DNA are between about 1 and 4000 μg, more typically about 1000 and 2000, most typically between about 2000 and 4000. [0052] The methods and compositions of the present invention are stimulate angiogenesis both in vitro and in vivo. When the endothelial cells to be stimulated are located within an animal, e.g., in an ischemic tissue or wound, the TEM gene will be administered to the animal in a pharmacologically acceptable form. Pharmaceutical compositions of the present invention will generally comprise an effective amount of the TEM protein or encoding DNA sequence composition dispersed in a pharmaceutically acceptable carrier or aqueous medium. As used herein, the phrases "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when a<iministered to an animal, or a human, as appropriate. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.

[0053] Various pharmaceutical compositions and techniques for their preparation and use will be known to those of skill in the art in light of the present disclosure. For a detailed listing of suitable pharmacological compositions and associated administrative techniques one may refer to the detailed teachings herein, which may be further supplemented by texts such as REMINGTON'S PHARMACEUTICAL SCIENCES, most recent edition, Mack Publishing Co.

[0054] The formulation and delivery methods will generally be adapted according to the site of desired angiogenesis and the disease to be treated. Exemplary formulations include, but are not limited to, those suitable for parenteral administration, e.g., intravenous, intraarterial, intramuscular, or subcutaneous administration, including formulations encapsulated in micelles, liposomes or drug-release capsules (active agents incorporated within a biocompatible coating designed for slow-release); ingestible formulations; formulations for topical use, such as creams, ointments and gels; and other formulations such as inhalants, aerosols and sprays. The dosage of the compounds of the invention will vary according to the extent and severity of the need for treatment, the activity of the administered composition, the general health of the subject, and other considerations well known to the skilled artisan.

[0055] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD₅o and ED₅o. Antibodies exhibiting high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition, age, weight, and therapeutic responsiveness. Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety sufficient to maintain the desired therapeutic effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data; for example, the concentration necessary to achieve 50-90% inhibition of migration activity using the assays described herein.

[0056] The mode of administration is not particularly important. In one embodiment, the mode of administration is an V. bolus. In another embodiment, the administration is topical. Alternately, one may administer the antibody in a local rather than systemic manner, for example, via injection of the antibody directly into a wound or an ischemic tissue, often in a depot or sustained release formulation.

[0057] Furthermore, one may administer the antibody in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody, targeting, for example, a wound or ischemic tissue. As the liposomes are carried in the blood and the present methods are particularly concerned with angiogenic behavior, individual liposome sizes are not considered to be a critical feature of the methods and compositions. Typically, intravenous injection of liposomal preparations are useful, but other routes of administration are also conceivable. The liposomes with the above formulations may be made still more specific for their intended targets with the incorporation of monoclonal antibodies or other ligands specific for a target. For example, monoclonal antibodies to the a specific receptor may be incorporated into the liposome by linkage to phosphatidylethanolamine (PE) incorporated into the liposome by the method of Leserman, L., et al, Nature (1980) 288:602-604. The liposomes will be targeted to and taken up selectively by the afflicted tissue.

[0058] The liposomes may be made from the present compositions in combination with any of the conventional synthetic or natural phospholipid liposome materials including phospholipids from natural sources such as egg, plant or animal sources such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin, phosphatidylserine, or phosphatidylinositol and the like. Synthetic phospholipids that may also be used, include, but are not limited to: dhnyristoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidycholine, and the corresponding synthetic phosphatidylethanolamines and phosphatidylglycerols. Cholesterol or other sterols, cholesterol hemisuccinate, glycolipids, cerebrosides, fatty acids, gangliosides, sphingolipids, l,2-bis(oleoyloxy)-3-(trimethyl ammonio) propane (DOTAP), N-[l-(2,3-dioleoyl) propyl-N,N,N-trimethylammonium chloride (DOTMA), and other cationic lipids may be incorporated into the liposomes, as is known to those skilled in the art. The relative amounts of phospholipid and additives used in the liposomes may be varied if desired. The preferred ranges are from about 60 to 90 mole percent of the phospholipid; cholesterol, cholesterol hemisuccinate, fatty acids or cationic lipids may be used in amounts ranging from 0 to 50 mole percent. The amounts of the present compounds incorporated into the lipid layer of liposomes can be varied with the concentration of the lipids ranging from about 0.01 to about 50 mole percent.

[0059] Other pharmaceutical formulations may also be used, dependent on the condition to be treated. For example, topical formulations are appropriate for treating pathological conditions such as wounds. As used herein, the term "topical" refers to topical to the wound, but is not limited to epidermal application. When applied topically, the TEM composition is usually combined with other ingredients, such as carriers and/or adjuvants. There are no limitations on the nature of such other ingredients, except that they must be pharmaceutically acceptable, efficacious for their intended administration, and cannot degrade or inactivate TEM composition. The TEM composition may be applied to burns in the form of an irrigant or salve, and if so then in an isotonic solution such as physiological saline solution or D5W. The TEM composition is particularly useful in accelerating the growth and survival of skin grafts applied to burns. Ordinarily, a TEM-containing composition is impregnated into the grafts or adherently coated onto the face of the graft, either on the side of the graft to be applied to the burn or on the exterior side of the graft. The TEM composition also is included in burn debridement salves that contain proteases so long as the debridement enzyme does not proteolytically inactivate the composition.

[0060] In the formulation of TEM compositions for topical use, creams, ointments and gels are also contemplated. The preparation of oleaginous or water-soluble ointment bases is also well known to those in the art. For example, these compositions may include vegetable oils, animal fats, and more preferably, semisolid hydrocarbons obtained from petroleum. Particular components used may include white ointment, yellow ointment, cetyl esters wax, oleic acid, olive oil, paraffin, petrolatum, white petrolatum, spermaceti, starch glycerite, white wax, yellow wax, lanolin, anhydrous lanolin and glyceryl monostearate. Various water-soluble ointment bases may also be used, including glycol ethers and derivatives, polyethylene glycols, polyoxyl 40 stearate and polysorbates. Even delivery through the skin may be employed if desired, e.g., by using transdermal patches, iontophoresis or electrotransport.

[0061] Pharmaceutical compositions for use in accordance with the present methods thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active TEM protein or DNA sequence into preparations that can be used pharmaceutically. These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0062] Proper formulation is dependent upon the route of administration chosen. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present methods.

[0063] The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0064] Pharmaceutical compositions comprising viral TEM DNA sequence vectors are dispersed in pharmacologically acceptable solutions or buffers. Preferred solutions include neutral saline solutions buffered with phosphate, lactate, Tris, and the like. The vector is sufficiently purified to render it essentially free of undesirable contaminant, such as defective interfering viral particles or endotoxins and other pyrogens such that it will not cause any untoward reactions in the individual receiving the vector construct. In one embodiment, purifying an adenovirus, involves the use of buoyant density gradients, such as cesium chloride gradient centrifugation. Supplementary active ingredients can also be incorporated into the compositions.

[0065] The vector containing the TEM-expressing DNA sequence can be any construct that permits the expression of the TEM protein, or a biologically active fragment thereof, in an infected cell. The manner of construct design useful in the present methods is conventional and is exemplified in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, F.M., et al, eds. 2000). Typically, the expression vectors include a promoter operably linked to the TEM DNA sequence, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences. Exogenous expression can be achieved using constitutive promoters, e.g., SV40, CMV, polyoma, adenovirus 2, promoter associated with genes that are expressed at high levels in mammalian cells such as elongation factor-1 or actin promoters, and the like, and inducible promoters known in the art. Further, it is also possible, and often desirable, to utilize endogenous promoter or control sequences, provided such control sequences are compatible with the host cell systems. Suitable promoters are any that permit TEM expression in the cell of interest, i.e., endothelial cells. The selection of appropriate promoters can readily be accomplished using conventional methods.

[0066] Viruses contemplated as useful vectors in the present methods and compositions include, but are not limited to lentiviruses, retroviruses, coxsackie viruses, herpes viruses (see, e.g., Geller, A. I. et al, ProcNatl. Acad. Scl: U.S.A. 90: 7603 (1993); Geller, A. I., et al, ProcNat. Acad. Sci USA 87:1149 (1990), adenoviruses (see, e.g., LaSalle et al, Science, 259:988 (1993); Davidson, et al, Nat. Genet 3: 219 (1993); Yang, et al, J. Virol. 69: 2004 (1995), adeno-associated viruses (see, e.g., Kaplitt, M. G., et al, Nat. Genet. 8:148 (1994))and the like.

[0067] In one embodiment, the vector is a replication-defective adenovirus. Techniques for preparing replication defective adenoviruses are well known in the art, as exemplified by Quantin, et al, Proc. Natl. Acad. Sci. USA, 89:2581-2584 (1992); Stratford-Perricadet, et al, J. Clin. Invest., 90:626-630 (1992); and Rosenfeld, et al, Cell, 68:143-155 (1992). h such an adenovirus, a viral gene essential for replication and/or packaging is deleted from the adenoviral vector construct, allowing the TEM expression region to be introduced in its place. Any gene, whether essential (e.g., El A, E1B, E2 and E4) or non-essential (e.g., E3) for replication, may be deleted and replaced with the TEM DNA sequence. Particularly preferred are those vectors and virions in which the El A and E1B regions of the adenovirus vector have been deleted and the TEM DNA sequence introduced in their place.

[0068] It is also well known that various cell lines may be used to propagate recombinant adenoviruses, so long as they complement any replication defect that may be present. One exemplary cell line is the human 293 cell line, but any other cell line that is permissive for replication, e.g., in the preferred case, which expresses El A and E1B may be employed. Further, the cells can be propagated either on plastic dishes or in suspension culture, in order to obtain virus stocks thereof. Other than the requirement that the adenovirus vector be replication defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. In one embodiment of the invention, a replication- defective, helper-independent adenovirus is created that expresses the TEM protein under the control of the human cytomegalovirus promoter.

[0069] For in vivo delivery of DNA, any suitable gene delivery system may be used including, e.g., viral- and liposome-mediated infection. As used herein, the term "infection" or "infected cell", is used to describe the targeted delivery of DNA to eukaryotic cells using delivery systems, such as, adenoviral, AAV, retroviral, or plasmid delivery gene transfer methods. Any suitable method may also be used to contact a cell with a TEM-encoding DNA sequence, so long as the method results in increased levels of functional TEM protein within the cell. The specificity of viral gene delivery may be selected to preferentially direct the gene to a particular target cell, such as by using viruses that are able to infect particular cell types. The introduction can be by standard techniques, e.g. infection, transfection, transduction or transformation. Examples of modes of gene transfer include e.g., naked DNA, CaPO₄ precipitation, DEAE dextran, electroporation, protoplast fusion, proteoliposomes, liposomes, cell microinjection, viral vectors and use of the "gene gun" as described, for example, by Fynan et al., Proc. Natl. Acad. Sci. U.S.A. 90: 11478-82 (1993).

[0070] The TEM DNA sequence compositions for use in the methods and compositions herein will often be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous or other such routes, particularly including direct instillation into a wound or ischemic disease site. The preparation of an aqueous composition that contains a TEM DNA sequence as an active ingredient will be known to those of skill in the art in light of the present disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.

[0071] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringabiUty exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0072] Recombinant adenovirus are often administered in amounts of between about 5x10⁹ and 5xl0¹² virus particles, which may be given either as a single bolus injection, as an intravenous infusion over several hours, or may be injected into an ischemic or wound site. It should also be pointed out that because the adenovirus vector employed in replication defective, it will not be capable of replicating in the cells that are ultimately infected. Moreover, it has been found that the genomic integration frequency of adenovirus is usually fairly low, typically on the order of about 1%. Thus, where continued treatment in certain individuals is required it may be necessary to reintroduce the virus every 6 months to a year.

[0073] The nucleic acid encoding the TEM protein can be administered to a blood vessel perfusing the ischemic tissue or to a site of vascular injury via a catheter, e.g.,, a hydrogel catheter, as described by U.S. Patent No. 5,652,225. The nucleic acid also can be delivered by injection directly into the ischemic tissue using the method described in U.S. Patent No. 6,121,246.

[0074] In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms are also contemplated, e.g., tablets or other solids for oral administration, time release capsules, liposomal forms and the like. Liposomal delivery of biological agents are exemplified in U.S. Pat. Nos. 4,485,054; 4,089,801; 4,234,871; and 4,016,100.

[0075] Optionally, therapeutically useful agents other than the TEM composition of the present methods also may be included in the pharmaceutical composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention. The TEM compositions can be administered alone or in combination with other pro- angiogenic agents in the form of proteins or nucleic acids. Such proteins include, e.g., acidic and basic fibroblast growth factors (aFGF and bFGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), transforming growth factor-α and β (TGF-α and TGF-β) platelet-derived endothelial growth factor (PD-ECGF), platelet-derived growth factor (PDGF), tumor necrosis factor α (TNF-α), hepatocyte growth factor (HGF), insulin like growth factor (IGF), erythropoietin, colony stimulating factor (CSF), macrophage-CSF (M-CSF), granulocyte/macrophage CSF (GM-CSF), nitric oxide synthase (NOS), L-arginine, fibronectin, urokinase, plasminogen activator and heparin. See, e.g., Klagsbrun, et al, Annu. Rev. Physiol, 53:217-239 (1991); Folkman, et al, J. Biol. Chem., 267:10931- 10934 (1992) and Symes, et al, Curr. Opin. Lipidology, 5:305-312 (1994).

[0076] The nucleotide sequence of such pro-angiogenic agents are readily available through a number of computer data bases, for example, GenBank, EMBL and Swiss-Prot. Using this information, a DNA segment encoding the desired may be chemically synthesized or, alternatively, such a DNA segment may be obtained using routine procedures in the art, e.g., PCR amplification, and co- administered with the TEM compositions.

[0077] The following examples are intended to illustrate but not to limit the invention.

Examples

Example 1 TEM 1

[0078] Adeno-TEM 1 Virus: The TEM expressing cells were produced using an adenovirus vector containing the gene encoding the full length TEM 1 (AD2CMV-TEM 1) constructed using the pAD (vantage) system as described in Souza et al, Biotechniques, 26; 502-08 (1999). The DNA sequence of TEM 1 is disclosed as SEQ ID NO. 196 in U.S. Serial No. 09/918715 (Publication No. 20030017157). AD2CMV-TEM 1 was used to infect human microvascular endothelial cells (HMVEC). The optimal multiplicity of infection for the adeno-TEM 1 virus in HMVECs was 300:1 (300 MOI) with an optimal time for protein expression being 67-72 hours. Seventy-two hours post-infection, HMVECs were placed in the proliferation assay. HMVECs infected with an empty vector (EV) or non-infected (NON) HMVECs served as the negative controls for the assay.

[0079] Proliferation Assay: Proliferation was assayed by the methods described in Crouch et al. , J. Immunol. Meth., 160; 81 (1993) and Kangas et al, Med. Biol. 62; 338 (1984). Briefly, proliferation was assessed in a 96 well plate system using 2xl0³ cells per well in media supplemented. The ATP luminescence assay (CellTiter Glo™ Luminescent Cell Viability kit, Promega) was then used to determine proliferation for the transfected and untransfected cells after 48 hours in various medias as indicated.

[0080] In cells grown in basal media, the HMVECs infected with empty vector (EV) and the non-infected (NON) cells produce less than 25,000 luminescence units after 48 hours. On the other hand, adeno-TEM infected HMVECs produced 190,000 luminescence units. Likewise, in the presence of 5% fetal bovine serum (FBS), adeno-TEM infected HMVECs proliferated to a greater degree than the EV- infected or the NON HMVECs. The adeno-TEM infected cells produced 350,000 luminescence units as compared to the 225,000 units and 230,000 luminescence units for EV infected and NON-HMVECs, respectively. In the presence of growth factor supplemented media, adeno-TEM infected HMVECs produced 490,000 luminescence units while the EV-infected and NON-HMVECs produced 350,000 luminescence units and 450,000 luminescence units, respectively. Thus, in all media, expression of TEM 1 stimulated the proliferation of HMVECs.

[0081] Migration Assay: Migration assay was performed as described in Glaser et al. , Nature, 288; 483-84 (1983) and Alessandria et al, Cancer Res. 43; 1790-97 (1983), using HMVECs 72 hours post-infection. Briefly, the HMVECs were plated in media without serum in the upper chamber an 8 micron pore membrane at 4x10 cells/chamber. Media supplemented with a chemo-attractant, 0.5% fetal bovine serum, was placed in the lower chamber. The HMVECs were allowed to migrate through to the lower side of the membrane over a 48 hour period. The cells that migrated through the membrane were then stained with Calcein AM (Molecular Probes) and quantified by fluorescence intensity.

[0082] Adeno-TEM 1 infection had no effect on the migration of HMVECs.

[0083] Endothelial Tube Formation Assay: Seventy-two hours post viral infection the HMVEC were trypsinized and plated onto to Matrigel™ for the tube formation assay. For the assay, HMVECs were plated at a density of 3 x 10⁴ cells/well in a 48 well plate with 250 μl of Matrigel™ per well. After 24 hours, HMVECs were stained with Calcein AM (Molecular Probes) for 30-60 minutes at 37°C, and fluorescence images of the tubes were captured using fluorescent microscopy. The area occupied by the tubes was quantified using MetaMorph image analysis.

[0084] In these experiments, HMVECs infected with the TEM 1 virus, but not the control virus, promoted and/or stabilized tube formation. The morphological changes evident in the adeno-TEM 1 infected cells were detectable as early as 2 hours after plating the infected HMVEC on Matrigel™. The increase and complexity of tube formation was observed after infection with the TEM 1 virus relative to the HMVEC infected with empty virus or the non-infected HMVEC.

Example 2 TEM 9

[0085] Adeno-TEM 9 Virus: The TEM9- expressing cells were produced using an adenovirus vector containing the gene encoding the full length TEM 9 (AD2CMV-TEM 1) constructed using the pAD (vantage) system as described in Souza et al, Biotechniques, 26; 502-08 (1999). The DNA sequence of TEM 9 is disclosed as SEQ ID NO. 212 in U.S. Serial No. 09/918715 (Publication No. 20030017157). AD2CMV-TEM 9 was used to infect human microvascular endothelial cells (HMVEC). The optimal multiplicity of infection for the adeno-TEM 9 virus in HMVECs was 300:1 (300 MOI) with an optimal time for protein expression being 67-72 hours. Seventy-two hours post-infection, HMVECs were placed in the proliferation assay. HMVECs infected with an empty vector (EV) or non-infected (NON) HMVECs served as the negative controls for the assay.

[0086] Proliferation Assay: Proliferation was assayed by the methods described in Crouch et al, J. Immunol. Meth., 160; 81 (1993) and Kangas et al, Med. Biol. 62; 338 (1984). Briefly, proliferation was assessed in a 96 well plate system using 2x10³ cells per well in media supplemented. The ATP luminescence assay (CellTiter Glo™ Luminescent Cell Viability kit, Promega) was then used to determine proliferation for the transfected and untransfected cells after 48 hours in various medias as indicated.

[0087] Exogenous expression of TEM 9 stimulated the proliferation of HMVECs. When incubated in basal media, adeno-TEM 9 infected HMVECs produced 175,000 luminescence units as compared to the EV-infected and NON-HMVECs that produced 50,000 and 60,000 luminescence units, respectively. In media supplemented with 5% FBS, the adeno-TEM infected HMVECs produced 325,000 luminescence units. The EV- and NON-HMVECs produced 150,000 and 170,000 luminescence units, respectively, in the FBS-supplemented media. In the growth factor supplemented media (GM media), the proliferation of the adeno-TEM 9 infected HMVECs again was greater than that of the HMVECs lacking exogenous TEM 9 expression, hi GM media, the adeno-TEM 9 infected HMVECs produced 365,000 luminescence units as compared to the 275,000 units and 280,000 luminescence units produced by the EV-infected and NON-HMVECs, respectively.

[0088] Migration Assay: Migration assay was performed as described in Glaser et al. , Nature, 288; 483-84 (1983) and Alessandria et al, Cancer Res. 43; 1790-97 (1983). Briefly, the HMVECs were plated in media without serum in the upper chamber an 8 micron pore membrane at 4x10⁴ cells/chamber. Media supplemented with a chemo-attractant, 0.5% fetal bovine serum, was placed in the lower chamber. The HMVECs were allowed to migrate through to the lower side of the membrane over a 48 hour period. The cells that migrated through the membrane were then stained with Calcein AM (Molecular Probes) and quantified by fluorescence intensity.

[0089] Exogenous TEM 9 expression increased endothelial cell migration, i.e., HMVECs had increased migration towards the fetal bovine serum when expressing exogenous TEM 9. Specifically, adeno-TEM infected HMVECs produced 2,393 fluorescence units as compared to 1,105 and 1,239 fluorescence units produce by the from the EV-infected and NON-HMVECs. Migrating NON-HMVEC cells produced only 135 fluorescent units in the absence of the chemo-attractant.

[0090] Endothelial Tube Formation Assay: Seventy-two hours post viral infection the HMVEC were trypsinized and plated onto to Matrigel™ for the tube formation assay. For the assay, HMVECs were plated at a density of 3 x 10⁴ cells/well in a 48 well plate with 250 μl of Matrigel™ per well. After 24 hours, HMVECs were stained with Calcein AM (Molecular Probes) for 30-60 minutes at 37°C, and fluorescence images of the tubes were captured using fluorescent microscopy. The area occupied by the tubes was quantified using MetaMorph image analysis.

[0091] Exogenous TEM 9 had no effect on tube formation of the infected HMVEC cells in the analysis performed.

Example 3 TEM 17

[0092] Adeno-TEM 17 Virus: The TEM 17- expressing HMVECs were produced using an adenovirus vector containing the gene encoding the full length TEM 17 (AD2CMV-TEM 1) constructed using the pAD (vantage) system as described in Souza et al, Biotechniques, 26; 502-08 (1999). The DNA sequence of TEM 17 is disclosed as SEQ ID NO. 230 in U.S. Serial No. 09/918715 (Publication No. 20030017157).AD2CMV-TEM 1 was used to infect human microvascular endothelial cells (HMVEC). The optimal multiplicity of infection for the adeno-TEM 9 virus in HMVECs was 300:1 (300 MOI) with an optimal time for protein expression being 67-72 hours. Seventy-two hours post- infection, HMVECs were placed in the proliferation assay. HMVECs infected with an empty vector (EV) or non-infected (NON) HMVECs served as the negative controls for the assay.

[0093] Proliferation Assay: Proliferation was assayed by the methods described in Crouch et al , J. Immunol. Meth., 160; 81 (1993) and Kangas et al, Med. Biol. 62; 338 (1984). Briefly, proliferation was assessed in a 96 well plate system using 2x10³ cells per well in media supplemented. The ATP luminescence assay (CellTiter Glo™ Luminescent Cell Viability kit, Promega) was then used to determine proliferation for the transfected and untransfected cells after 48 hours in various medias as indicated.

[0094] Exogenous TEM 17 expression did not stimulate HMVEC proliferation in this assay.

Migration Assay: Migration assay was performed as described in Glaser et al, Nature, 288; 483- 84 (1983) and Alessandria et al, Cancer Res. 43; 1790-97 (1983). Briefly, the HMVECs were plated in media without serum in the upper chamber an 8 micron pore membrane at 4x10⁴ cells/chamber. Media supplemented with a chemo-attractant, 0.5% fetal bovine serum, was placed in the lower chamber. The HMVECs were allowed to migrate tlirough to the lower side of the membrane over a 48 hour period. The cells that migrated through the membrane were then stained with Calcein AM (Molecular Probes) and quantified by fluorescence intensity.

[0095] The infection of HMVEC with adeno-TEM 17 did not increase migration of proliferation of the HMVEC.

F00961 Endothelial Tube Formation Assay: Seventy-two hours post viral infection the HMVEC were trypsinized and plated onto to Matrigel™ for the tube formation assay. For the assay, HMVECs were plated at a density of 3 x 10⁴ cells/well in a 48 well plate with 250 μl of Matrigel™ per well. After 24 hours, HMVECs were stained with Calcein AM (Molecular Probes) for 30-60 minutes at 37°C, and fluorescence images of the tubes were captured using fluorescent microscopy. The area occupied by the tubes was quantified using MetaMorph image analysis.

[0097] Exogenous TEM 17 expression promotes and/or stabilizes tube formation of HMVECs when HMVECs are grown in HCTl 16 conditioned media. The greater complexity of the tubule formation was a parent based on visual inspection. The increased complexity of tubule formation was confirmed using MetaMorph image analysis. In basal media, the tube area for the adeno-TEM 17-infected HMVECs was 18,000 while EV-infected HMVECs had a tube area of only 11 ,000. The NON-HMVEC also had a tube area of 18,000 in the presence of basal media, i the presence of HCTl 16 colon carcinoma-conditioned media, the adeno-TEM 17-infected HMVECs had a tube area of 27,500 while the EV-infected HMVECs had a tube area of approximately 11,000. The NON-HMVECs also had a lower tube area relative to the adeno-TEM 17-infected HMVECs with a tube area of 24,000. The greater tube area for the adeno- TEM 17 infected HMVEC was also observed with HT29 colon carcinoma-conditioned media, hi the presence of this media, adeno-TEM 17-infected HMVECs had a tube area of 25,000. The EV-infected HMVECs had a tube area of 10,000 and the NON-HMVECs had a tube are of 21,000. In GM media, the adeno-TEM 17-infected HMVECs had a tube area of 27,500 while the EV-infected HMVECs had a tube area of 20,000 and the NON-HMVECs had a tube area of 28,500. These experiments demonstrate the ability of exogenous TEM 17 expression to stimulate endothelial tube formation of HMVECs in the presence of conditioned media.

[0098] Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, yet these modifications and improvements are within the scope and spirit of the invention, as set forth in the claims which follow.

[0099] Citation of the above publications or documents is not intended as an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. U.S. patents and other publications referenced herein are hereby incorporated by reference.

Claims

Claims

1. A method of stimulating endothelial cell proliferation, comprising administering to an endothelial cell, an effective amount of a (tumor endothelial marker) TEM 1 protein, or a biologically active protein fragment thereof, whereby the endothelial cell is stimulated to proliferate.

2. The method of claim 1 , wherein the TEM protein is administered as a vector comprising a nucleic acid encoding the TEM protein, or a biologically active protein fragment thereof.

3. The method of claim 2, wherein said vector is a viral vector.

4. The method of claim 3, wherein said vector is a replication deficient adenovirus.

5. A method of stimulating endothelial cell proliferation, comprising administering to an endothelial cell, an effective amount of a (tumor endothelial marker) TEM 9 protein, or a biologically active protein fragment thereof, whereby the endothelial cell is stimulated to proliferate.

6. The method of claim 5, wherein the TEM protein is administered as a vector comprising a nucleic acid encoding the TEM protein, or a biologically active protein fragment thereof.

7. The method of claim 6, wherein said vector is a viral vector.

8. The method of claim 7, wherein said vector is a replication deficient adenovirus.

9. A method of stimulating endothelial cell migration, comprising administering to an endothelial cell, an effective amount of a (tumor endothelial marker) TEM 9 protein, or a biologically active protein fragment thereof, whereby the endothelial cell is stimulated to migrate.

10. The method of claim 9, wherein the TEM protein is administered as a vector comprising a nucleic acid encoding the TEM protein, or a biologically active protein fragment thereof.

11. The method of claim 10, wherein said vector is a viral vector.

12. The method of claim 11, wherein said vector is a replication deficient adenovirus.

13. A method of stimulating endothelial cell tubule formation, comprising administering to an endothelial cell, an effective amount of a (tumor endothelial marker) TEM 1 protein, or a biologically active protein fragment thereof, whereby the endothelial cell is stimulated to form tubules.

14. The method of claim 13, wherein the TEM protein is administered as a vector comprising a nucleic acid encoding the TEM protein, or a biologically active protein fragment thereof.

15. The method of claim 14, wherem said vector is a viral vector.

16. The method of claim 15, wherein said vector is a replication deficient adenovirus.

17. A method of stimulating endothelial cell tubule formation, comprising administering to an endothelial cell, an effective amount of a (tumor endothelial marker) TEM 17 protein, or a biologically active protein fragment thereof, whereby the endothelial cell is stimulated to form tubules.

18. The method of claim 17, wherein the TEM protein is administered as a vector comprising a nucleic acid encoding the TEM protein, or a biologically active protein fragment thereof.

19. The method of claim 18 , wherein said vector is a viral vector.

20. The method of claim 19, wherein said vector is a replication deficient adenovirus.

21. The method of claim 17, further comprising administering colon carcinoma-conditioned media.

22. A recombinant virus construct comprising a DNA sequence encoding tumor endothelial marker (TEM) 1, or a biologically active fragment thereof.

23. The virus of claim 22, wherein the virus is a replication deficient adenovirus.

24. A recombinant virus construct comprising a DNA sequence encoding tumor endothelial marker (TEM) 9, or a biologically active fragment thereof.

25. The virus of claim 24, wherein the virus is a replication deficient adenovirus.

26. A recombinant virus construct comprising a DNA sequence encoding tumor endothelial marker (TEM) 17, or a biologically active fragment thereof.

27. The virus of claim 26, wherein the virus is a replication deficient adenovirus.

28. A method of stimulating angiogenesis, comprising administering to a subject in need thereof, an effective amount of a (tumor endothelial marker) TEM 1 protein, or a biologically active protein fragment thereof, and angiogenesis is stimulated.

29. The method of claim 28, wherein the TEM protein is administered as a vector comprising a nucleic acid encoding the TEM protein, or a biologically active protein fragment thereof.

30. The method of claim 29, wherein said vector is a viral vector.

31. The method of claim 30, wherein said vector is a replication deficient adenovirus.

32. The method of claim 28, wherein the subject has anoxic tissue damage.

33. The method of claim 28, wherem the subject has chronic wound healing deficiencies.

34. The method of claim 28, wherein infection of endothelial cells with said vector results in increased proliferation and increased tube formation.

35. A method of stimulating angiogenesis, comprising administering to a subject in need thereof, an effective amount of a TEM 9 protein, or a biologically active protein fragment, and angiogenesis is stimulated.

36. The method of claim 35, wherein the TEM protein is administered as a vector comprising a nucleic acid encoding the TEM protein, or a biologically active protein fragment thereof

37. The method of claim 36, wherein said vector is a viral vector.

38. The method of claim 37, wherein said vector is a replication deficient adenovirus.

39. The method of claim 35, wherein the subject has anoxic tissue damage.

40. The method of claim 35, wherein the subject has chronic wound healing deficiencies.

41. The method of claim 35 wherein infection of endothelial cells with said vector results in increased proliferation and migration.

42. A method of stimulating angiogenesis, comprising administering to a subject in need thereof, an effective amount of a TEM 17 protein, or a biologically active protein fragment, and angiogenesis is stimulated.

43. The method of claim 42, wherein the TEM protein is administered as a vector comprising a nucleic acid encoding the TEM protein, or a biologically active protein fragment thereof.

44. The method of claim 43, wherein said vector is a viral vector.

45. The method of claim 44, wherein said vector is a replication deficient adenovirus.

46. The method of claim 42 wherein the subject has anoxic tissue damage.

47. The method of claim 42, wherein the subject has chronic wound healing deficiencies.

48. The method of claim 42, wherein infection of endothelial cells with said vector results in increased tube formation.