Compositions and methods for inhibiting angiogenesis

#Blood vessels are constructed by two processes: vasculogenesis, whereby a primitive vascular network is established during embryogenesis from multipotential mesenchymal progenitors; and angiogenesis, in which preexisting vessels send out capillary sprouts to produce new vessels. Endothelial cells are centrally involved in each process. They migrate, proliferate and then assemble into tubes with tight cell-cell connections to contain the blood (Hanahan, Science 277:48-50 (1997)). Angiogenesis occurs when enzymes, released by endothelial cells, and leukocytes begin to erode the basement membrane, which surrounds the endothelial cells, allowing the endothelial cells to protrude through the membrane. These endothelial cells then begin to migrate in response to angiogenic stimuli, forming off-shoots of the blood vessels, and continue to proliferate until the off-shoots merge with each other to form the new vessels.

#Normally angiogenesis occurs in humans and animals in a very limited set of circumstances, such as embryonic development, wound healing, and formation of the corpus luteum, endometrium and placenta. However, aberrant angiogenesis is associated with a number of disorders, including, tumor metastasis. In fact, it is commonly believed that tumor growth is dependent upon angiogenic processes. Thus, the ability to increase or decrease angiogenesis has significant implications for clinical situations, such as wound healing (e.g., graft survival) or cancer therapy, respectively.

#Antithrombin or Antithrombin III (AT3) is a single chain glycoprotein involved in the coagulation process. It is synthesized primarily in the liver with a signal peptide of 32 amino acids necessary for its intracellular transport through the endoplasmic reticulum; the peptide is then cleaved prior to secretion. Mourey et al., Biochimie 72:599-608 (1990).

#FIG. 1a. shows three conformations of AT3. b. (SEQ ID NO: 1) shows the amino acid sequence of human AT3 and the pancreatic elastase cleavage site of the c-terminal reactive loop.

#FIG. 2 shows the sequence data and schematic of bovine AT3 and aaAT3. N-terminal sequences were determined by automated Edman degradation on a PE/ABD Procise 494cLC protein sequencer (Foster City, Calif.) with high sensitivity phenylthiohydantoin amino acid detection by capillary HPLC. Sequence library searches and alignments were performed against combined GenBank, Brookhaven Protein, SWISS-PROT, and PIR databases.

#FIG. 3 is a graph showing the effects of intact (S), cleaved (R) and (L) conformations of bovine AT3 on capillary endothelial cell proliferation.

#As previously discussed, aberrant angiogenesis is associated with a number of disorders including tumor metastasis. Additionally, endothelial cells are associated with angiogenesis. In the present invention, fragments, conformations, biological equivalents, or derivatives of AT3 may exhibit anti-angiogenic and anti-tumor activity. For example, the R and L forms of AT3 are endothelial cell specific. Thus, this invention provides for methods of reducing or inhibiting angiogenesis, tumor growth and endothelial proliferations using fragments, conformations, biological equivalents, or derivatives of AT3. The invention also provides for methods of identifying inhibitors of tumor growth, endothelial cell proliferation, and/or angiogenesis. The invention also provides for pharmaceutical compositions comprising a fragment, conformation, biological equivalent, or derivative of AT3. The elements of the invention will now be discussed.

#AT3 can be derived from any organism which produces the protein in nature. In a particular embodiment the organism is bovine or human. The amino acid sequence of bovine AT3 is available under GenBank Accession No. 1168462, and the amino acid sequence of human AT3 is available under GenBank Accession No. 113936 (SEQ ID NO:1).

#AT3 can be isolated from body fluids such as serum, ascites and urine. AT3 can also be synthesized chemically or biologically, such as by cell culture or recombinant technology, or produced transgenically. Similarly, the particular portions and conformations of AT3, which are the subject of this invention, can be isolated from natural sources, produced transgenically, or can be chemically or biologically synthesized, such as by guanidine treatment or in vitro cleavage of AT3. Recombinant techniques known in the art include, but are not limited to, gene amplification from DNA using polymerase chain reaction (PCR), gene amplification from RNA using reverse transcriptase PCR and NASBA (nucleic acid sequence based amplifications).