Novel Gene And Protein Associated With Angiogenesis And Endothelial Cell Specific Apoptosis

Abstract

This invention provides isolated nucleic acid and amino acid sequences encoding VG5Q, a novel angiogenic growth factor protein with pro-angiogenic activity, a forkhead-associated domain, a G-patch domain; characteristic subcellular localization in an in vitro Matrigel model of angiogenesis: towards the cell periphery in early stages of tubulogenesis, between cells in newly formed endothelial tubes, and no nuclear staining after 24 hours; is expressed in endothelial cells; is secreted during angiogenesis; and interacts with TWEAK. The invention also provides for expression vectors containing nucleic acid sequences encoding VG5Q protein, and host cells containing one or more expression vectors for the recombinant expression of VG5Q. The invention also provides for methods of using VG5Q for the diagnosis and treatment of angiogenesis-mediated diseases or disorders.

1 . An isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of:
 (a) a nucleic acid encoding a VG5Q protein, which protein has pro-angiogenic activity, a forkhead-associated domain, a G-patch domain; characteristic subcellular localization in an in vitro Matrigel model of angiogenesis: towards the cell periphery in early stages of tubulogenesis, between cells in newly formed endothelial tubes, and no nuclear staining after 24 hours; is expressed in endothelial cells; is secreted during angiogenesis; and interacts with TWEAK;   (b) a nucleic acid sequence coding for a polypeptide with the amino acid sequence of SEQ ID NO:2;   (c) a nucleic acid comprising the sequence shown in SEQ ID NO: 1;   (d) a nucleic acid sequence coding for a polypeptide with the amino acid sequence of SEQ ID NO:4;   (e) a nucleic acid comprising the sequence shown in SEQ ID NO:3; and   (f) a nucleic acid sequence complementary to the sequence of (a), (b), (c), (d) or (e).   
     
     
         2 - 7 . (canceled) 
     
     
         8 . A nucleic acid sequence consisting of at least 15 contiguous nucleotides of a nucleic acid sequence of  claim 1  and variants thereof. 
     
     
         9 - 10 . (canceled) 
     
     
         11 . A vector comprising the nucleic acid described in  claim 1 . 
     
     
         12 . An isolated host cell comprising the vector of  claim 11 . 
     
     
         13 . A method of producing VG5Q protein, which method comprises culturing the host cell of  claim 12  under conditions such that the VG5Q protein is expressed. 
     
     
         14 . A method of producing cells expressing VG5Q protein, which method comprises introducing the vector of  claim 11  into cells. 
     
     
         15 . An isolated VG5Q protein which protein has pro-angiogenic activity, a forkhead associated domain, a G-patch domain; characteristic subcellular localization in an in vitro Matrigel model of angiogenesis: towards the cell periphery in early stages of tubulogenesis, between cells in newly formed endothelial tubes, and no nuclear staining after 24 hours; is expressed in endothelial cells; is secreted during angiogenesis; and interacts with TWEAK. 
     
     
         16 . The isolated protein of  claim 15  comprising an amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence shown in SEQ ID NO: 4, an amino acid sequence having 90% sequence identity to SEQ ID NO: 2 or an amino acid sequence having 90% identity to SEQ ID NO: 4. 
     
     
         17 - 19 . (canceled) 
     
     
         20 . A nucleic acid delivery vehicle for inducing angiogenesis in a mammal, the nucleic acid delivery vehicle comprising a nucleic acid comprising the isolated nucleic acid of  claim 1 , and further comprising a nucleic acid delivery carrier. 
     
     
         21 - 23 . (canceled) 
     
     
         24 . The nucleic acid delivery vehicle  claim 20 , wherein the nucleic acid delivery carrier is i) a vector selected from the group consisting of a plasmid, a retrovirus, an adenovirus, and a virus; ii) a lipid compound; or iii) a gene gun. 
     
     
         25 . (canceled) 
     
     
         26 . The nucleic acid delivery vehicle of  claim 24 , wherein the lipid compound is selected from the group consisting of liposomes, lipofectins, cytofectins, and lipid-based positive ions. 
     
     
         27 . (canceled) 
     
     
         28 . A method of enhancing and/or inducing the expression of VG5Q in a mammal comprising administering the nucleic acid delivery vehicle of  claim 20  to target cells of the subject. 
     
     
         29 . A method of treating diseases characterized by insufficient angiogenesis in a mammal by the method of  claim 28 . 
     
     
         30 . The nucleic acid of  claim 8 , which nucleic acid comprises an anti-sense nucleic acid sequence a siRNA, or a ribozyme directed to a portion of the nucleic acid for inhibiting and/or suppressing the expression of VG5Q in a mammal. 
     
     
         31 - 32 . (canceled) 
     
     
         33 . A method of inhibiting and/or suppressing the expression of VG5Q in a mammal comprising administering the nucleic acid of  claim 30  to the mammal. 
     
     
         34 - 37 . (canceled) 
     
     
         38 . A nucleic acid delivery vehicle for inhibiting angiogenesis in a mammal, the nucleic acid delivery vehicle comprising a nucleic acid comprising the isolated nucleic acid of  claim 30 , and further comprising a nucleic acid delivery carrier. 
     
     
         39 . (canceled) 
     
     
         40 . A peptide fragment of a polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4. 
     
     
         41 . The peptide fragment of  claim 40  comprising an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51. 
     
     
         42 . An antibody which binds to the peptide fragment in  claim 40 . 
     
     
         43 - 44 . (canceled) 
     
     
         45 . The purified antibody of  claim 42  that inhibits the binding of VG5Q protein to its receptor. 
     
     
         46 . A purified antibody of  claim 42  that selectively binds to an epitope in the receptor-binding domain of VG5Q protein. 
     
     
         47 . The antibody of  claim 46 , wherein the epitope is within the sequence in SEQ ID NO: 7, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 58. 
     
     
         48 - 50 . (canceled) 
     
     
         51 . A method of inhibiting the action of VG5Q in a subject comprising administering the antibody of  claim 42 . 
     
     
         52 . A therapeutic composition to reduce or suppress angiogenesis in a mammal, the therapeutic composition comprising a therapeutically effective amount of the antibody of  claim 45  and a pharmaceutically acceptable carrier. 
     
     
         53 . A method of treating a disease or disorder in a subject characterized by excessive angiogenesis comprising administering an effective amount of the therapeutic composition of  claim 52  to the mammal. 
     
     
         54 . (canceled) 
     
     
         55 . A therapeutic composition to enhance and/or induce angiogenesis in a mammal, the therapeutic composition comprising a therapeutically effective amount of an isolated protein, which protein has pro-angiogenic activity, a forkhead-associated domain, a G-patch domain; characteristic subcellular localization in an in vitro Matrigel model of angiogenesis: towards the cell periphery in early stages of tubulogenesis, between cells in newly formed endothelial tubes, and no nuclear staining after 24 hours; is expressed in endothelial cells; is secreted during angiogenesis; and interacts with TWEAK. 
     
     
         56 . The composition of  claim 55 , wherein the protein is encoded by an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4. 
     
     
         57 . (canceled) 
     
     
         58 . A method of treating a disease or disorder in a mammal characterized by insufficient angiogenesis comprising administering the therapeutic composition of  claim 55  to the mammal. 
     
     
         59 . An isolated DNA encoding a mutant VG5Q protein, wherein the wild-type protein has pro-angiogenic activity, a forkhead-associated domain, a G-patch domain; characteristic subcellular localization in an in vitro Matrigel model of angiogenesis:
 towards the cell periphery in early stages of tubulogenesis, between cells in newly formed endothelial tubes, and no nuclear staining after 24 hours; is expressed in endothelial cells; is secreted during angiogenesis; and interacts with TWEAK.   
     
     
         60 . The DNA of  claim 59 , wherein said DNA comprises SEQ ID NO: 1 with a mutation, an A at base 688. 
     
     
         61 - 64 . (canceled) 
     
     
         65 . The DNA of  claim 59 , wherein said DNA comprises a mutation altering an arginine at position 133 of SEQ ID NO: 2. 
     
     
         66 - 68 . (canceled) 
     
     
         69 . A method of expressing a gene in endothelial cells comprising introducing into cells a construct comprising the nucleic acid of SEQ ID NO: 8 operably linked to the coding sequence of a gene. 
     
     
         70 . A method of inducing and/or enhancing angiogenesis in a subject in need of such angiogenesis comprising the administration of an isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of:
 (a) a nucleic acid encoding a VG5Q protein, which protein has pro-angiogenic activity, a forkhead-associated domain, a G-patch domain; characteristic subcellular localization in an in vitro Matrigel model of angiogenesis: towards the cell periphery in early stages of tubulogenesis, between cells in newly formed endothelial tubes, and no nuclear staining after 24 hours; is expressed in endothelial cells; is secreted during angiogenesis; and interacts with TWEAK;   (b) a nucleic acid sequence coding for a polypeptide with the amino acid sequence of SEQ ID NO:2;   (c) a nucleic acid comprising the sequence shown in SEQ ID NO: 1;   (d) a nucleic acid sequence coding for a polypeptide with the amino acid sequence of SEQ ID NO:4;   (e) a nucleic acid comprising the sequence shown in SEQ ID NO:3; and   (f) a nucleic acid sequence complementary to the sequence of (a), (b), (c), (d) or (e)   
       or an isolated VG5Q protein which has pro-angiogenic activity, a forkhead associated domain, a G-patch domain; characteristic subcellular localization in an in vitro Matrigel model of angiogenesis: towards the cell periphery in early stages of tubulogenesis, between cells in newly formed endothelial tubes, and no nuclear staining after 24 hours; is expressed in endothelial cells; is secreted during angiogenesis; and interacts with TWEAK. 
     
     
         75 - 77 . (canceled) 
     
     
         78 . A method of inhibiting and/or suppressing angiogenesis in a subject in need of such treatment comprising administering a an isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of:
 (a) a nucleic acid encoding a VG5Q protein, which protein has pro-angiogenic activity, a forkhead-associated domain, a G-patch domain; characteristic subcellular localization in an in vitro Matrigel model of angiogenesis: towards the cell periphery in early stages of tubulogenesis, between cells in newly formed endothelial tubes, and no nuclear staining after 24 hours; is expressed in endothelial cells; is secreted during angiogenesis; and interacts with TWEAK;   (b) a nucleic acid sequence coding for a polypeptide with the amino acid sequence of SEQ ID NO:2;   (c) a nucleic acid comprising the sequence shown in SEQ ID NO: 1;   (d) a nucleic acid sequence coding for a polypeptide with the amino acid sequence of SEQ ID NO:4;   (e) a nucleic acid comprising the sequence shown in SEQ ID NO:3; and   (f) a nucleic acid sequence complementary to the sequence of (a), (b), (c), (d) or (e)   
       or an isolated VG5Q protein which has pro-angiogenic activity, a forkhead associated domain, a G-patch domain; characteristic subcellular localization in an in vitro Matrigel model of angiogenesis: towards the cell periphery in early stages of tubulogenesis, between cells in newly formed endothelial tubes, and no nuclear staining after 24 hours; is expressed in endothelial cells; is secreted during angiogenesis; and interacts with TWEAK. 
     
     
         79 . (canceled)