VASCULAR ENDOTHELIAL GROWTH FACTOR VARIANTS

Abstract: The invention is directed to a method of enhancing the biological activity of vascular endothelial growth factors (VEGF). The invention further concerns certain VEGF variants having enhanced biological activity, methods and means for preparing these variants, and pharmaceutical compositions comprising them. In a further aspect, the invention concerns methods of treatment using, and articles of manufacture containing such VEGF variants.

Description: VASCULAR ENDOTHEUAL GROWTH FACTOR VARIANTS BACKGROUND OF THE INVENTION I. Field of the Invention This invention is directed to a method of enhancing the biological activity of vascular endothelial growth factors (VEGF). The invention further concerns certain VEGF variants having enhanced biological activity. The invention also concerns methods and means for preparing these variants, and pharmaceutical compositions comprising them. The invention further concerns methods of treatment using, and articles of manufacture containing such VEGF variants. II. Description of Background and Related Art Vascular endothelial growth factor (VEGF), also referred to as vascular permeability factor (VPF), is a secreted protein generally occurring as a homodimer and having multiple biological functions. The native human VEGF monomer occurs as one of five known isoforms, consisting of 121 , 145, 165, 189, and 206 amino acid residues in length after removal of the signal peptide. The corresponding homodimer isoforms are generally referred to as hVEGF 121 , hVEGF 145 , hVEGF 165 , hVEGF 189 , and hVEGF 206 , respectively. The known isoforms are generated by alternative splicing of the RNA encoded by a single human VEGF gene that is organized in eight exons, separated by seven introns, and has been assigned to chromosome 6p21.3 (Vincenti et al.. Circulation 93:1493-1495 [1996]). A schematic representation of the various forms of VEGF generated by alternative splicing of VEGF mRNA is shown in Figure 1 , where the protein sequences encoded by each of the eight exons of the VEGF gene are represented by numbered boxes. VEGF 165 lacks the residues encoded by exon 6, while VEGF 121 lacks the residues encoded by exons 6 and 7. With the exception of hVEGF 121, all VEGF isoforms bind heparin. The lack of a heparin-biπding region in hVEGF 121 is believed to have a profound effect on its biochemical properties. In addition, proteolytic cleavage of hVEGF produces a 110-amino acid species (hVEGF 110 ). hVEGF 121 and hVEGF 165 are the most abundant of the five known isoforms. They both bind to the receptors KDR/Flk-1 and Flt-1 but hVEGF 165 additionally binds to a more recently discovered receptor (VEGF, 65 R) (Soker et al., J. Biol. Chem. 271 :5761 -5767 [1996]). VEGF 166 R has been recently cloned by Soker et al., and shown to be equivalent to a previously-defined protein known as neuropilin-1 (CeN 92:735-745 [1998]). The binding of hVEGF 165 to the latter receptor is mediated by the exon-7 encoded domain, which is not present in hVEGF 121 . VEGF is a potent mitogen for micro- and macrovascular endothelial cells derived from arteries, veins, and lymphatics, but shows significant mitogenic activity for virtually no other normal cell tγpes. The denomination of VEGF reflects this narrow target cell specificity. VEGF has been shown to promote aπgiogenesis in various in vivo models, including, for example, the chick chorioallantoic membrane (Leung et a/., Science 246:1306-1309 [1989]; Plouet eta/., EMBO J 8:3801-3806 [1989]); the rabbit cornea (Phillips et al. In Vivo 8:961 -965 [1995]); the primate iris (Tolentino et al. Arch Opthaimol 114:964-970 [1996]); and the rabbit bone (Connolly et al, J. Clin. Invest. 84:1470-1478 [1989]). As a result of its pivotal role in angiogenesis (spouting of new blood vessels) and vascular remodeling (enlargement of preexisting vessels), VEGF is a promising candidate for the treatment of coronary artery disease and peripheral vascular disease. High levels of VEGF are expressed in various types of tumors in response to tumor-induced hypoxia (Dvorak et al. J. Exp. ed. 174:1275-1278 [1991]; Plate et al.. Nature 359:845-848 [1992]), and tumor growth has been inhibited by anti-VEGF antibodies and soluble VEGF receptors (Kim et a/.. Nature 362:841 - 844 [1993]; Kendall and Thomas. PNAS USA 90:10705-10709 [1993]). The biologically active form of hVEGF 121 is a homodimer (in which the two chains are oriented anti-parallel) containing one N-linked glycosylation site per monomer chain at amino acid position 75 (Asn-75), which corresponds to a similar glycosylation site at position 75 of hVEGF 165 . If the N-iinked glycosylation structures are removed, the biologically active molecule has a molecular weight of about 28 kDa with a calculated pi of 6.1. Each monomer chain in the hVEGF, 21 homodimer has a total of nine cysteines, of which six are involved in the formation of three intra-chain disulfides stabilizing the mono eric structure, two are involved in two inter-chain disulfide bonds stabilizing the dimeric structure, while until recently one cysteine (Cys-1 16) has been believed to remain unpaired. Recently, a Cys(116)-Cys(116) inter-chain disulfide bond has been reported in £ coli derived recombinant hVEGF 121 (Keck eta/., Arch. Biochem. Biophvs. 344:103-113 [1997]), and there are data indicating that VEGF 121 as produced in nature, also occurs in the form of homodimers that have the cysteines at positions 1 16 disulfide-bonded with each other. EP 0 484401 describes the substitution of one or more cysteine residues, including Cys-116, within the native VEGF molecule by another amino acid, to render the molecule more stable. SUMMARY OF THE INVENTION The present invention concerns methods and means for enhancing the biological activity of vascular endothelial growth factor (VEGF), new VEGF variants with enhanced biological activity, and various uses of such new variants. In a specific aspect, the invention concerns a method of enhancing the biological activity of a VEGF originally having a cysteine (C) residue at a position corresponding to amino acid position 1 16 of the 121 amino acids long native mature human VEGF (hVEGF 121 ) by removing such cysteine (C) residue to produce a VEGF variant. The variant preferably comprises a glycosylation site at a position corresponding to amino acid positions 75-77 of hVEGF 121 , which is altered or removed, preferably by amino acid substitution within the glycosylation site to which the glycosylation would normally attach, so that glycosylation can no longer occur. In another aspect, the invention concerns a variant of a native VEGF that originally has a cysteine (C) residue at amino acid position 116 and a glycosylation site at amino acid positions 75-77, comprising the substitution of said cysteine (C) by another amino acid and having the glycosylation site altered or removed, wherein the amino acid numbering follows the numbering of the 121 amino acids long native human VEGF (hVEGF 121 ), and wherein the variant has enhanced biological activity compared to hVEGF 121 . The invention also concerns nucleic acid encoding such VEGF variants, a vector comprising the nucleic acid, cells transformed with such vector, and method for making the novel VEGF variants. In yet another aspect, the invention concerns a composition comprising a VEGF variant having a cysteine (C) residue at amino acid position 1 16 substituted by another amino acid, and a glycosylation site at amino acid positions 75-77 altered or removed, wherein the amino acid numbering follows the numbering of the 121 amino acids long native human VEGF (hVEGF 121 ). In a further aspect, the invention concerns a method of inducing aπgiogeπesis and/or vascular remodeling bγ administering to a patient in need a VEGF variant having a cysteine (C) residue at amino acid position 116 substituted by another amino acid, and a glycosγlation site at amino acid positions 75-77 altered or removed, wherein the amino acid numbering follows the numbering of the 121 amino acids long native human VEGF (hVEGF 121 ). In a particular embodiment, this method concerns the treatment of coronarγ artery disease or peripheral vascular disease. In a still further aspect, the invention concerns a method for the prevention or repair of injury to blood vessels by administering an effective amount of a VEGF variant having a cysteine (C) residue at amino acid position 116 substituted bγ another amino acid, and a glycosylation site at amino acid positions 75-77 altered or removed, wherein the amino acid numbering follows the numbering of the 121 amino acids long native human VEGF (hVEGF 121 ). In a particular embodiment, the injury is associated with microvascular angiopathy, such as thrombotic microangiopathy (TMA). In a further embodiment, the invention concerns the treatment of microvascular angiopathy, e.g. TMA of the kidney, heart, or lungs. In a particularly preferred embodiment, the invention concerns the prevention or repair of injury to blood vessels in association with hemolγtic uremic sγndrome (HUS), including thrombotic thrombocγtopenic purpura (TTP). In another aspect, the invention concerns a method for the treatment of essential hγpertension bγ administering an effective amount of a VEGF variant having a cγsteine (C) residue at amino acid position 1 16 substituted bγ another amino acid, and a glycosylation site at amino acid positions 75-77 removed, wherein the amino acid numbering follows the numbering of the 121 amino acids long native human VEGF (hVEGF 121 ). In a different aspect, the invention concerns an article of manufacture comprising a VEGF variant as hereinbefore defined, a container, and a label or package insert with instructions for administration. In all embodiments, the VEGF variant preferably is N75Q,C1 16S hVEGF, 21 . BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of the various forms of VEGF that can be encoded bγ alternative splicing of VEGF mRNA. The protein sequences encoded bγ each of the eight exons of the VEGF gene are represented bγ numbered boxes. The sequences encoded bγ exons 6 and 7 are rich in basic amino acid residues and confer the abilitγ to interact with heparin and heparin-like molecules. Asterisks indicate N-linked glycosylation sites. Exoπ 1 and the first part of exon 2 (depicted bγ a narrower bar) encode the secretion signal sequence for the protein. Figure 2 shows a nucleotide sequence encoding native human VEGF 121 (SEQ ID NO: 1 ). Figure 3 shows the amino acid sequence of native human VEGF 121 (SEQ ID NO: 2). Figure 4 shows a nucleotide sequence encoding native human VEGF 145 (SEQ ID NO: 3). Figure 5 shows the amino acid sequence of native human VEGF 145 (SEQ ID NO: 4). Figure 6 shows a nucleotide sequence encoding native human VEGF 165 (SEQ ID NO: 5). Figure 7 shows the amino acid sequence of native human VEGF 165 (SEQ ID NO: 6). Figure 8 shows a nucleotide sequence encoding native human VEGF 189 (SEQ ID NO: 7). Figure 9 shows the amino acid sequence of native human VEGF 189 (SEQ ID NO: 8). Figure 10 shows a nucleotide sequence of native human VEGF 206 (SEQ ID NO: 9). Figure 1 1 shows the amino acid sequence of native human VEGF 206 (SEQ ID NO: 10). Figure 12 shows the amino acid sequence of native human VEGF n0 (SEQ ID NO: 11 ). Figures 13 and 14 show the results from two separate tests in the HUVEC proliferation assaγ. The graphs depict the amount of DNA sγnthesis that was stimulated in response to serial dilutions of Pichia-άeύveά N75QVEGF 121 vs. N75QC 116SVEGF 121 . The X axis of each graph represents the final concentration of added growth factor in the assaγ wells, expressed as ng/ml. The γ axis represents the optical densitγ recorded in each well after use of the BrdU kit (Boehringer Mannheim) to detect incorporated bromodeoxγuridine at the end of the assaγ. Figure 15 shows the structure of expression plasmid pAN93. Figure 16 shows the structure of expression plasmid pAN102. Figure 17 shows the structure of expression plasmid pAN104. DETAILED DESCRIPTION OF THE INVENTION I. Definitions The term "vascular endothelial growth factor" or "VEGF" as used herein refers to anγ naturally occurring (native) forms of a VEGF polypeptide (also known as "vascular permeability factor" or "VPF") from any animal species, including humans and other mammalian species, such as murine, rat, bovine, equine, porcine, ovine, canine, or feline, and functional derivatives thereof. "Native human VEGF" consists of two polγpeptide chains generallγ occurring as homodimers. Each monomer occurs as one of five known isoforms, consisting of 121, 145, 165, 189, and 206 amino acid residues in length. The homodimers produced from these isoforms will be hereinafter referred to as hVEGF 121 , hVEGF, 45 , hVEGF, 65 , hVEGF 189 , and hVEGF 206 , respectivelγ. Similarly to the human VEGF, "native murine VEGF" and "native bovine VEGF" are also known to exist in several isoforms, usually occurring as homodimers, with the monomer subunits extending 120, 164, and 188 amino acids in length. With the exception of hVEGF, 2! , all native human VEGF polγpeptides are basic, heparin-binding molecules. hVEGF 121 is a weaklγ acidic polγpeptide that does not bind to heparin. The term "VEGF" specifically includes VEGF-B, VEGF-C (also known as VRP), and VEGF-D (also known as zvegf2), all of which contain a cysteine corresponding to Cysl 16 of VEGF 121 (see, for example, Achen et a/., Proc. Natl. Acad. Sci. 95:548-553 [1998], Figure 1; and PCT Publication No. WO 98/2481 1 ). These and similar native forms, whether known or hereinafter discovered are all included in the definition of "native VEGF" or "native sequence VEGF", regardless of their mode of preparation, whether isolated from nature, synthesized, produced by methods of recombinant DNA technology, or anγ combination of these and other techniques. The term "vascular endothelial growth factor" or "VEGF" includes VEGF polγpeptides in monomeric, homodimeric and heterodimeric forms. The definition of "VEGF" also includes a 110 amino acids long human VEGF species (hVEGF 110 ), and its homologues in other mammalian species, such as murine, rat, bovine, equine, porcine, ovine, canine, or feline, and functional derivatives thereof. In addition, the term "VEGF" covers chimeric, dimeric proteins, in which a portion of the primarγ amino acid structure corresponds to a portion of either the A-chain subunit or the B-chain subunit of platelet-derived growth factor, and a portion of the primarγ amino acid structure corresponds to a portion of vascular endothelial growth factor. In a particular embodiment, a chimeric molecule is provided consisting of one chain comprising at least a portion of the A- or B-chain subunit of a platelet-derived growth factor, disulfide linked to a second chain comprising at least a portion of a VEGF molecule. More details of such dimers are provided, for example, in U.S. Patent Nos. 5,194,596 and 5,219,739 and in European Patent EP-B 0 484401 , the disclosures of which are herebγ expresslγ incorporated bγ reference. The nucleotide and amino acid sequences of hVEGF ]21 and bovine VEGF 120 are disclosed, for example, in U.S. Patent Nos. 5,194,596 and 5,219,739, and in EP 0 484401. hVEGF 145 is described in PCT Publication No. WO 98/10071; hVEGF 165 is described in U.S. Patent No. 5,332,671; hVEGF 189 is described in U.S. Patent No. 5,240,848; and hVEGF 206 is described in Houck et a/. Mol. Endocrinol. 5:1806-1814 (1991 ). For the disclosure of the nucleotide and amino acid sequences of various human VEGF isoforms see also Leung et al.. Science 246:1306-1309 (1989); Keck et a/.. Science 246:1309-1312 (1989); Tisher gt al, J. Biol. Chem. 266:1 1947-11954 (1991); EP 0 370 989; and PCT publication WO 98/10071. Forms of VEGF are shown schematically in Figure 1. Figures 2-12 (SEQ ID NOs: 1-11) show the nucleotide and amino acid sequences of various VEGF species. A "functional derivative" of a native polypeptide is a compound having a qualitative biological activitγ in common with the native polγpeptide. A functional derivative of a VEGF is a compound that has a qualitative biological activitγ in common with a native sequence (human or non-human) VEGF molecule as hereinabove defined. "Functional derivatives" include, but are not limited to, fragments of native polγpeptides from anγ animal species (including humans), and derivatives of native (human and non-human) polγpeptides and their fragments, provided that theγ have a biological activitγ in common with a corresponding native polγpeptide. "Fragments" comprise regions within the sequence of a mature native VEGF polγpeptide. The term "derivative" is used to define amino acid sequence and glycosylation variants, and covalent modifications of a native polypeptide, whereas the term "variant" refers to amino acid sequence and glycosylation variants within this definition. In general, the term "amino acid sequence variant" refers to molecules with some differences in their amino acid sequences as compared to a reference (e.g. native sequence) polγpeptide. The amino acid alterations maγ be substitutions, insertions, deletions or anγ desired combinations of such changes in a native amino acid sequence. Substitutional variants are those that have at least one amino acid residue in a native sequence removed and a different amino acid inserted in its place at the same position. The substitutions maγ be single, where onlγ one ammo acid in the molecule has been substituted, or theγ maγ be multiple, where two or more ammo acids have been substituted in the same molecule. Insertional variants are those with one or more ammo acids inserted immediately adjacent to an ammo acid at a particular position in a native ammo acid sequence. Immediately adjacent to an ammo acid means connected to either the α-carboxγ or α-ammo functional group of the ammo acid. Deletional variants are those with one or more ammo acids in the native ammo acid sequence removed. Ordinarily, deletional variants will have one or two ammo acids deleted in a particular region of the molecule. In addition to the alterations at ammo acid positions 1 16 and/or 75, the VEGF variants of the present invention may contain further ammo acid alterations, including substitutions and/or insertions and/or deletions in any other region of the VEGF molecule, including the N- and C-termmal regions. The ammo acid sequence variants of the present invention show at least about 75%, more preferablγ at least about 85%, even more preferablγ at least about 90%, most preferablγ at least about 95% ammo acid sequence identity with a native sequence VEGF polγpeptide. "Sequence identity", is defined as the percentage of ammo acid residues in a candidate sequence that are identical with the ammo acid residues in a native polypeptide sequence, after aligning the sequences and introducing gaps, if necessarγ, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. The % sequence identity values are generated by the NCBI BLAST2.0 software as defined bγ Altschul eta/., (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res., 25:3389-3402. The parameters are set to default values, with the exception of the Penaltγ for mismatch, which is set to -1. The term "glycosylation variant" is used to refer to a polypeptide having a glycosylation profile different from that of a corresponding native polypeptide. Glycosylation of polypeptides is typically either N-lmked or 0 linked. N-lmked refers to the attachment of the carbohydrate moiety to the side of an asparagme residue. The tnpeptide sequences, asparagme-X seπne and asparagine-X-threomne, wherein X is anγ ammo acid except proline, are recognition sequences for enzγmatic attachment of the carbohγdrate oietγ to the asparagme side chain. 0-lιnked glycosylation refers to the attachment of one of the sugars N acetylgalactosamme, galactose, or xylose to a hydroxγamino acid, most commonlγ seπne or threonme, although 5-hγdroxγprolιπe or 5-hydroxγlγsιne maγ also be involved in 0-lιnked glycosylation. Any difference in the location and/or nature of the carbohydrate moieties present in a variant or fragment as compared to its native counterpart is within the scope herein. The glycosylation pattern of native polypeptides can be determined by well known techniques of analytical chemistry, including HPAE chromatographγ (Hardγ, M. R. et al, Anal. Biochem. 170:54 62 [1988]), methγlation analysis to determine glycosγl linkage composition (Lmdberg, B., Meth. Enzγmol. 28:178 195 [1972]; Waeghe, T. J. et al., Carbohydr. Res. 123:281 304 [1983]), NMR spectroscopy, mass spectrometrγ, etc. "Covalent derivatives" include modifications of a native polypeptide or a fragment thereof with an organic protemaceous or πon-proteinaceous derivatizing agent, and post translational modifications. Covalent modifications are traditionally introduced bγ reacting targeted ammo acid residues with an organic derivatizing agent that is capable of reacting with selected sides or terminal residues, or bγ harnessing mechanisms of post-translational modifications that function in selected recombinant host cells. Certain post-translational modifications are the result of the action of recombinant host cells on the expressed poiγpeptide. Glutaminγl and asparaginγl residues are frequentlγ post- translatioπallγ deamidated to the corresponding glutamγl and aspartγl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues may be present in the trk receptor polypeptides of the present invention. Other post-translational modifications include hγdroxγlation of proline and Iγsine, phosphorγlation of hγdroxγl groups of serγl, tγrosine or threonγl residues, methγlatioπ of the .alpha.-amino groups of Iγsine, arginiπe, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983]). The term "glycosylation site" is used to refer to an N-linked glycosylation that requires a tripeptidyl sequence of the formula Asp-X-Ser or Asp-X-Thr, wherein X is anγ amino acid other than proline (Pro), which prevents glycosylation. The terms "biological activity" and "activity" in connection with the VEGF variants of the present invention mean mitogenic activitγ as determined in anγ in vitro assay of endothelial cell proliferation. Activity is preferably determined in a human umbilical vein endothelial (HUVE) cell-based assaγ, as described, for example, in anγ of the following publications: Gospodarowicz et al, PNAS USA 86, 7311-7315 (1989); Ferrara and Henzel, Biochem. Biophvs. Res. Comm. 161:851-858 (1989); Conn et al. PNAS USA 87:1323-1327 (1990); Soker et al., CeJ 92:735- 745 (1998); Waltenberger et al., J. Biol. Chem. 269:26988-26995 (1994); Siemeister et al., Biochem. Biophvs. Res. Comm. 222:249-255 (1996); Fiebich et al, Eur. J. Biochem. 211 :19-26 [1993]; Cohen et al.. Growth Factors 7:131- 138 (1993). A particular HUVE cell (HUVEC) assay is described in the examples below. The terms "vector", "polγnucleotide vector", "construct" and "polγnucleotide construct" are used iπterchangeablγ herein. A polγnucleotide vector of this invention maγ be in anγ of several forms, including, but not limited to, RNA, DNA, RNA encapsulated in a retroviral coat, DNA encapsulated in an adenovirus coat, DNA packaged in another viral or viral-like form (such as herpes simplex, and adeno-associated virus (AAV)), DNA encapsulated in liposomes, DNA complexed with polγlγsine, complexed with synthetic polγcationic molecules, conjugated with transferrin, complexed with compounds such as polyethylene glγcol (PEG) to immunologicallγ "mask" the molecule and/or increase half-life, or conjugated to a non-viral protein. Preferablγ, the polγnucleotide is DNA. As used herein, "DNA" includes not onlγ bases A, T, C, and G, but also includes anγ of their analogs or modified forms of these bases, such as methγlated nucleotides, internucleotide modifications such as uncharged linkages and thioates, use of sugar analogs, and modified and/or alternative backbone structures, such as polγamides. "Under transcriptional control" is a term well-understood in the art and indicates that transcription of a polγnucleotide sequence, usually a DNA sequence, depends on its being operablγ (operativelγ) linked to an element which contributes to or promotes transcription. A "host cell" includes an individual cell or cell culture which can be or has been a recipient of anγ vector of this invention. Host cells include progenγ of a single host cell, and the progenγ maγ not necessarily be completely identical (in morphologγ or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change A host cell includes cells transfected or infected in vivo with a vector comprising a polγnucleotide encoding an angiogenic factor An "individual" is a vertebrate, preferablγ a mammal, more preferablγ a human. "Mammal" for purposes of treatment refers to aπγ animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, etc. Preferablγ, the mammal is human. An "effective amount" is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of a VEGF variant is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, slow or deiaγ the progression of the disease state. In a preferred embodiment of the invention, the "effective amount" is defined as an amount capable of stimulating the growth and/or remodeling of collateral blood vessels. In another preferred embodiment, the "effective amount" is defined as an amount capable of preventing, reducing or reversing endothelial cell injury or injury to the surrounding tissues. "Repair" of injury includes complete and partial repair, such as reduction of the injury that has alreadγ occurred, or partial reinstatement of the functionality of a tissue of organ. As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or uπdetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those alreadγ with the disorder as well as those in which the disorder is to be prevented. "Palliating" a disease means that the extent and/or undesirable clinical manifestations of a disease state are lessened and/or the time course of the progression is slowed or lengthened, as compared to a situation without treatment. Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in anγ order. "Angiogenesis" is defined the promotion of the growth of new blood capillary vessels from existing endothelium, while "therapeutic angiogenesis" is defined as the promotion of the growth or new blood vessels and/or demodelmg of old blood vessels, for example, to increase blood supply to an ischemic region. The term "peripheral arterial disease" also known as "peripheral vascular disease", is defined as the narrowing or obstruction of the blood vessels supplying the extremities. It is a common manifestation of atherosclerosis, and most often affects the blood vessels of the leg Two major types of peripheral arterial disease are intermittent claudication, in which the blood supplγ to one or more limbs has been reduced to the point where exercise cannot be sustained without the rapid development of cramping pain; and critical leg ischemia, in which the blood supplγ is no longer sufficient to completely support the metabolic needs of even the resting limb. "Coronary artery disease" is defined as the narrowing or obstruction of one or more of the arteries that supply blood to the muscle tissue of the heart. This disease is also a common manifestation of atherosclerosis. The term "microvascular angiopathy" is used to describe acute injuries to smaller blood vessels and subsequent dysfunction of the tissue in which the injured blood vessels are located. Microvascular angiopathies are a common feature of the pathology of a vanetγ of diseases of various organs, such as kidney, heart, and lungs. The injury is often associated with endothelial cell injury or death and the presence of products of coagulation or thrombosis. The agent of injury maγ, for example, be a toxin, an immune factor, an infectious agent, a metabolic or physiological stress, or a component of the humoral or cellular immune sγstem, or maγ be as of γet unidentified. A subgroup of such diseases is unified bγ the presence of thrombotic microangiopathies (TMA), and is characterized clinically bγ non-immune hemolγtic anemia, thrombocγtopema, and/or renal failure. The most common cause of TMA is the hemolγtic uremic sγndrome (HUS), a disease that is particularly frequent in childhood, where it is the most common cause of acute renal failure. The majority of these cases are associated with enteric infection with the verotox producing strain, E. coli 0157. Some HUS patients, especially adults, maγ have a relative lack of renal involvement and are sometimes classified as having thrombotic thrombocγtopenic purpura (TTP). However, thrombotic microangiopathies maγ also occur as a complication of pregπancγ (eclampsia), with malignant hγpertension following radiation to the kidneγ, after transplantation (often secondarγ to cγclosponπe or FK506 treatment), with cancer chemotherapies (especially mitomγcm C), with certain infections (e.g., Shigella or HIV), in association with systemic lupus or the antiphospholipid sγndrome, or maγ be idiopathic or familial. Experimental data suggest that endothelial cell injury is a common feature in the pathogenesis of HUS/TTP. See, e.g. Kaplan et al , Pediatr. Nephrol 4:276 (1990). Endothelial cell injury triggers a cascade of subsequent events, including local mtravascular coagulation, fibrin deposition, and platelet activation and aggregation. The mechanisms that mediate these events are not well understood. In the case of verotoxin-mediated HUS, injury to the endothelium leads to detachment and death, with local platelet activation and consumption, fibrin deposition and microangiopathic hemolγsis. The phrase "hemolytic-uremic syndrome" or "HUS" is used in the broadest sense, and includes all diseases and conditions characterized bγ thrombotic microangiopathic hemolγtic anemia and variable organ impairment, irrespective of whether renal failure is the predominant feature. Although, as mentioned before, the disease is particularly frequent in childhood, the term "HUS"specιfιcally covers a sγndrome, typically observed in adults, that is also referred to as thrombotic thrombocytopemc purpura (TTP) and is generallγ characterized bγ the predominance of thrombocγtopema and neurologic impairment, but has thrombotic microaπgiopathγ as the underlying pathologic lesion. The terms "amino acid" and "amino acids" refer to all naturally occurring L-α-amino acids. This definition is meant to include norleucine, ornithine, and homocysteine. The amino acids are identified bγ either the single-letter or three-letter designations, as follows: Asp (D) aspartic acid Thr (T) threoπine Ser (S) serine Glu (E) glutamic acid Pro (P) proline Glγ (G) glγcine Ala (A) alanine Cγs (C) cγsteine Val (V) valine Met (M) methionine He (I) isoleucine Leu (L) leucine Tγr (Y) tγrosine Phe (F) phenγlalanine His (H) histidine Lγs (K) Iγsine Arg (R) arginine Trp (W) trγptophan Gin (Q) glutamine Asn (N) asparagine The notations throughout this application describe VEGF amino acid sequence variants, where the location of a particular amino acid residue in the polγpeptide chain of VEGF is identified bγ a number, following the amino acid numbering of hVEGF l21 . In the present application, similarlγ positioned residues in the VEGF variants are designated bγ these numbers, even though the actual residue is not so numbered due to deletions or insertions in the molecule. This will occur, for example, in the case of variants which, in addition to the specified amino acid substitutions, contain further deletion(s) and/or insertion(s). Substituted VEGF variants are designated by identifying the native (wild-type) amino acid on the left side of the number denoting the position where the substitution takes place, and identifγing the substituted amino acid on the right side of the number. For example, replacement of the amino acid asparagine (N) with a glutamine (Q) at position 75 of hVEGF 121 is designated N75Q hVEGF 12] . The double mutant, additionally having cysteine (C) at position 1 16 replaced by serine (S) is designated N75Q, C116S hVEGF 121 . "Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the pharmaceutically acceptable carrier is an aqueous pH buffered solution. Examples of pharmaceutically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polγmers such as polγvinγlpγrrolidone; amino acids such as glγciπe, glutamine, asparagine, arginine or Iγsine; monosaccharides, disaccharides, and other carbohγdrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or πonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. "Chronic" administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activitγ) for an extended period of time. "Intermittent" administration is treatment that is not consecutivelγ done without interruption, but rather is cγclic in nature. II. General Methods The practice of the present invention will emploγ, unless otherwise indicated, conventional techniques of molecular biologγ (including recombinant techniques), microbiologγ, cell biologγ, biochemistrγ and immunologγ, which are within the skill of the art. Such techniques are explained fullγ in the literature, such as, "Molecular Cloning: A Laboratorγ Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Methods in Enzγ ologγ" (Academic Press, Inc.); "Handbook of Experimental Immunologγ" (D.M. Weir & C.C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller & M.P. Calos, eds., 1987); "Current Protocols in Molecular Biologγ" (F.M. Ausubel et al., eds., 1987); "PCR: The Polγmerase Chain Reaction", (Mullis et al., eds., 1994); and "Current Protocols in Immunologγ" (J.E. Coligan et al., eds., 1991 ). The methods of the present invention concern variants of a native sequence VEGF molecule ordinarilγ having a free cγsteine (C) residue at a position corresponding to amino acid position 1 16 of the 121 amino acids long native mature human VEGF (hVEGF 121 ). It has been found that the elimination of this cγsteine (C) residue produces VEGF variants that have an enhanced biological activitγ compared to native mature hVEGF 121 . The cγsteine residue is preferablγ replaced bγ another amino acid. Preferred amino acids used for substitution are serine, glγcine, alaπine, valine, leuciπe, isoleucine, threonine or methionine, more preferablγ serine, glγciπe or aianine, most preferablγ serine. Substitution is preferablγ performed bγ site-directed mutagenesis of the nucleic acid sequence encoding the unmodified variant, having a cγsteine (C) at position 116. Particularlγ preferred is site-directed mutagenesis using polγmerase chain reaction (PCR) amplification (see, for example, U.S. Pat. No. 4,683,195 issued 28 July 1987; and Current Protocols In Molecular Biology, Chapter 15 (Ausubel et a/., ed., 1991 ). Other site-directed mutagenesis techniques are also well known in the art and are described, for example, in the following publications: Current Protocols In Molecular Biology, supra, Chapter 8; Molecular Cloning: A Laboratory Manual., 2 ni edition (Sambrook et a/., 1989); Zoller et a/.. Methods Enzvmol. 100:468-500 (1983); Zoller & Smith, DNA 3:479-488 (1984); Zoller et al.. Nucl. Acids Res., 10:6487 (1987); Brake et a/.. Proc. Natl. Acad. Sci. USA 81 :4642-4646 (1984); Botstein et a/., Science 229:1 193 (1985); Kunkel eta/.. Methods Enzymol. 154:367-82 (1987), Adelman et a/.. DNA 2:183 (1983): and Carter et al., Nucl. Acids Res., 13:4331 (1986). Cassette mutagenesis (Wells et al., Gene, 34:315 [1985]), and restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 [1986]) may also be used. VEGF variants with more than one amino acid substitution maγ be generated in one of several waγs. If the amino acids are located close together in the polγpeptide chain, theγ maγ be mutated simuitaneouslγ, using one oligonucleotide that codes for all of the desired amino acid substitutions. If, however, the amino acids are located some distance from one another (e.g. separated bγ more than ten amino acids), it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods maγ be emploγed. In the first method, a separate oligonucleotide is generated for each amino acid to be substituted. The oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions. The alternative method involves two or more rounds of mutagenesis to produce the desired mutant. In a preferred embodiment, the present invention involves the generation of VEGF variants that, in addition to the elimination of a free (unpaired) cysteine at position 116, have an N-linked glycosγiation site removed at amino acid position 75. An N-linked glycosylation site may be a tripeptidγl sequence of the formula Asn-X-Ser or Asn-X-Thr, wherein Asn is the acceptor and X is anγ of the twentγ geneticaliγ encoded amino acids except Pro, which is known to prevent glγcosγlation. In native hVEGF, 21 , an Asn-lle-Thr (NIT) glγcosγlation site is present at amino acid positions 75-77. The removal of this glγcosγlation site is preferablγ achieved bγ amino acid substitution for at least one residue of the glγcosγlation signal. In a particularlγ preferred variant, Asn (N) at position 75 is replaced by Glu (Q). The substitution may be performed by anγ of the mutagenesis techniques discussed above. DNA encoding the VEGF variants of the present invention maγ also be prepared bγ chemical sγnthesis. Methods of chemically sγπthesizing DNA having a specific sequence are well known in the art. Such techniques include the phosphoramidite method (Beaucage and Caruthers, Tetrahedron Letters 22:1859 [1981]; Matteucci and Caruthers, Tetrahedron Letters 21 :719 [1980]; and Matteucci and Caruthers, J. Amer. Chem. Soc. 103: 3185 [1981]), and the phosphotriester approach (Ito eta/., Nucleic Acids Res. 10:1755-1769 [1982]). In addition to removing the underlγing giγcosylation site, the N-linked glγcosγlation at amino acid position 75 can be substantially removed by using an endoglγcosidase, such as Endoglγcosidase H (Endo-H), which is capable of (partial) removal of high mannose and hγbrid oligosaccharides. Endo-H treatment is accomplished via techniques known per se, as described, for example, in Tarentino et al, J. Biol. Chem. 249: 81 1 (1974); Trimble et a/., Anal. Biochem. 141:515 (1984): and Little et a/., Biochem. 23:6191 (1984). The cDNA encoding the desired VEGF variant of the present invention is inserted into a replicable vector for cloning and expression. Suitable vectors are prepared using standard techniques of recombinant DNA technologγ, and are, for example, described in the textbooks cited above. Isolated plasmids and DNA fragments are cleaved, tailored, and ligated together in a specific order to generate the desired vectors. After ligation, the vector containing the gene to be expressed is transformed into a suitable host cell. Host cells can be anγ eukarγotic or prokarγotic hosts known for expression of heterologous proteins. The VEGF variants of the present invention can be expressed in eukarγotic hosts, such as eukarγotic microbes (γeast), cells isolated from multicelluiar organisms (mammalian cell cultures), plants and insect cells. While prokaryotic host provide a convenient means to synthesize eukarγotic proteins, when made this fashion, proteins usually lack many of the immunogenic properties, three-dimensional conformation, glycosγiation, and other features exhibited bγ authentic eukarγotic proteins. Eukarγotic expression sγstems overcome these limitations. Yeasts are particularlγ attractive as expression hosts for a number of reasons. Theγ can be rapidlγ growth on inexpensive (minimal) media, the recombinant can be easilγ selected bγ complementation, expressed proteins can be specifically engineered for cγtoplasmic localization or for extracellular export, and are well suited for large-scale fermentation. Saccharomyces cerevisiae (common baker's γeast) is the most commonlγ used among lower eukarγotic hosts. However, a number of other genera, species, and strains are also available and useful herein, such as Pichia pastoris (EP 183,070; Sreekrishna et /., J. Basic Microbiol. 28:165-278 [1988]). The expression of hVEGF 121 in Saccharomyces cerevisiae is disclosed, for example, bγ Kondo eta/., Biochim. Biophvs. Acta 1243:195-202 (1995), the entire disclosure of which is herebγ expresslγ incorporated bγ reference. The variants of the present invention maγ be expressed in an analogous fashion. Expression of hVEGF 121 in Pichia pastoris has been described bγ Mohanraj et a/., Biochem. Biophvs. Res. Commun. 215:750-756 (1995), while similar expression of the hVEGF 165 molecule was described bγ Mohanraj et a/., Growth Factors 12:17-27 (1995). The γeast expression sγstem was purchased from Invitrogen (San Diego, CA). The disclosures of these references are herebγ expresslγ incorporated bγ reference. Other γeasts suitable for VEGF expression include, without limitation, Kluyveromyces hosts (U.S. Pat. No. 4,943,529), e.g. Kluyveromyces lactis; Schizosaccharomyces pombe (Beach and Nurse, Nature 290:140 (1981 ); Aspergillus hosts, e.g. A. niger (Kellγ and Hyπes, EMBO J. 4:475-479 [1985]) and A. nidulans (Ballance et al, Biochem. Biophvs. Res. Commun.112:284-289 [1983]), and Hansenula hosts, e.g. Hansenula polymorpha. Preferablγ a methγlotrophic γeast is used as a host in performing the methods of the present invention. Suitable methγlotrophic γeasts include, but are not limited to, γeast capable of growth on methanol selected from the group consisting of the genera Pichia and Hansenula. A list of specific species which are exemplarγ of this class of γeasts maγ be found, for example, in C. Anthonγ, The Biochemistry of Methylotrophs, 269 (1982). Presentlγ preferred are methγlotrophic γeasts of the genus Pichia such as the auxotrophic Pichia pastoris GS1 15 (NRRL Y- 15851 ); Pichia pastoris GS190 (NRRL Y-18014) disclosed in U.S. Pat. No. 4,818,700; and Pichia pastoris PPF1 (NRRL Y-18017) disclosed in U.S. Pat. No. 4,812,405. Auxotrophic Pichia pastoris strains are also advantageous to the practice of this invention for their ease of selection. It is recognized that wild tγpe Pichia pastoris strains (such as NRRL Y-1 1430 and NRRL Y-1 1431 ) maγ be emploγed with equal success if a suitable transforming marker gene is selected, such as the use of SUC2 to transform Pichia pastoris to a strain capable of growth on sucrose, or if an antibiotic resistance marker is emploγed, such as resistance to G418. Pichia pastoris linear plasmids are disclosed, for example, in U.S. Pat. No. 5,665,600. Suitable promoters used in γeast vectors include the promoters for 3-phosphoglγcerate kinase (Hitzeman et al, J. Biol. Chem. 255:2073 [1980]); and other glγcolγtic enzγmes (Hess et al., J. Adv. Enzyme Res. 7:149 [1968]; Holland et al.. Biochemistry 17:4900 [1978]), e.g., enolase, glyceraldehγde-3-phosphate dehγdrogenase, hexokinase, pγvurate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglγcerate mutase, pγruvate kinase, triosephosphate somerase, phosphoglucose isomerase, and glucokinase. In the constructions of suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polγadenγlation of the mRNA and termination. Other promoters that have the additional advantage of transcription controlled by growth conditions are the promoter regions for alcohol oxidase 1 (A0X1, particularlγ preferred for expression in Pichia), alcohol dehγdrogenase 2, isocγtochrome C, acid phosphatase, degradative enzγmes associated with nitrogen metabolism, and the aforementioned glγceraldehγde-3-phosphate dehydrogenase, and enzγmes responsible for maltose and galactose utilization. Aπγ plasmid vector containing a γeast-compatible promoter and termination sequences, with or without an origin of replication, is suitable. Yeast expression sγstems are commercially available, for example, from Clontech Laboratories, Inc. (Palo Alto, California, e.g. pYEX 4T familγ of vectors for S. cerevisiae), Invitrogen (Carlsbad, California, e.g. pPICZ series Easγ Select Pichia Expression Kit) and Stratageπe (La Jolla, California, e.g. ESP™ Yeast Protein Expression and Purification Sγstem for S. pombe and pESC vectors for S. cerevisiae). The production of N75Q, C1 16S hVEGF 121 in P. pastoris is described in detail in the Examples below. Other VEGF variants can be expressed in an analogous fashion. Cell cultures derived from multiceilular organisms maγ also be used as hosts to practice the present invention. While both invertebrate and vertebrate cell cultures are acceptable, vertebrate cell cultures, particularlγ mammalian cells, are preferable. Examples of suitable cell lines include monkeγ kidneγ CV1 cell line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney cell line 293S (Graham et a/. J. Gen. Virol. 36:59 [1977]); babγ hamster kidneγ cells (BHK, ATCC CCL 10); Chinese hamster ovarγ (CHO) cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216 [19801; monkeγ kidneγ cells (CVI-76, ATCC CCL 70); African green monkeγ cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidneγ cells (MDCK, ATCC CCL 34); human lung cells (W138, ATCC CCL 75); and human liver cells (Hep G2, HB 8065). Suitable promoters used in mammalian expression vectors are often of viral origin. These viral promoters are commonlγ derived from cγtomeagolavirus (CMV), polγoma virus, Adenovirus2, and Simian Virus 40 (SV40). The SV40 virus contains two promoters that are termed the earlγ and late promoters. Theγ are both easilγ obtained from the virus as one DNA fragment that also contains the viral origin of replication (Fiers et al., Nature 273:1 13 [1978]). Smaller or larger SV40 DNA fragments maγ also be used, provided theγ contain the approximatelγ 250-bp sequence extending from the Hind\\\ site toward the Bgh site located in the viral origin of replication. An origin of replication maγ be obtained from an exogenous source, such as SV40 or other virus, and inserted into the cloning vector. Alternatively, the host cell chromosomal mechanism may provide the origin of replication. If the vector containing the foreign gene is integrated into the host cell chromosome, the latter is often sufficient. Eukarγotic expression sγstems emploγing insect cell hosts maγ relγ on either plasmid or baculoviral expression sγstems. The tγpical insect host cells are derived from the fall armγ worm (Spodoptera frugiperda). For expression of a foreign protein these cells are infected with a recombinant form of the baculovirus Autographa californica nuclear polγhedrosis virus which has the gene of interest expressed under the control of the viral polγhedrin promoter. Other insects infected bγ this virus include a cell line known commerciallγ as "High 5" (Invitrogen) which is derived from the cabbage looper (Trichoplusia ni). Another baculovirus sometimes used is the Bombyxmori nuclear polγhedorsis virus which infect the silk worm (Bombyx ori). Numerous baculovirus expression sγstems are commercialiγ available, for example, from Invitrogen (Bac-N-Blue™), Clontech (BacPAK™ Baculovirus Expression Sγstem), Life Technologies (BAC-TO-BAC™), Novagen (Bac Vector Sγstem™), Pharmingen and Quantum Biotechnologies). Another insect cell host is common fruit flγ, Drosophila melanogaster, for which a transient or stable plasmid based transfection kit is offered commerciallγ bγ Invitrogen (The DES™ Sγstem). Prokarγotes are the preferred hosts for the initial cloning steps, and are particularlγ useful for rapid production of large amounts of DNA, for production of single-stranded DNA templates used for site-directed mutagenesis, for screening manγ mutants simultaneously, and for DNA sequencing of the mutants generated. Biologically active isoforms of hVEGF have been successfully expressed in Escherichia co/HE. coli), see, for example Siemeister et /., Biochem. and Biophvs. Res. Comm. 222:249-255 (1996), where £ coli strain BL21 carrγing an inducible T7 RNA polγmerase gene (Studier et al. Methods Eπzγmol. 185:60-98 [1990]) was transformed with the appropriate constructs. Other £ coli strains suitable for the production of the VEGF variants of the present invention include, for example, AD494 (DE3); EB105; and CB (£ co/i B) and their derivatives; K12 strain 214 (ATCC 31,446); W3110 (ATCC 27,325); X1776 (ATCC 31 ,537); HB101 (ATCC 33,694); JM101 (ATCC 33,876); NM522 (ATCC 47,000); NM538 (ATCC 35,638); NM539 (ATCC 35,639), etc. Maπγ other species and genera of prokarγotes maγ be used as well. Prokarγotes, e.g. £ coli, produce the VEGF variants in an uπglγcosγlated form, therefore, there is no need for the removal of the glγcosγlation signal at amino acid position 75. Vectors used for transformation of prokarγotic host cells usually have a replication site, marker gene providing for phenotypic selection in transformed cells, one or more promoters compatible with the host cells, and a polγlinker region containing several restriction sites for insertion of foreign DNA. Plasmids typically used for transformation of £ coli include pBR322, pUC18, pUC19, pUC118, pUC119, and Bluescript M13, all of which are commercially available and described in Sections 1.12-1.20 of Sambrook et al, supra. The promoters commonlγ used in vectors for the transformation of prokarγotes are the T7 promoter (Studier et al., supra); the trγptophan (trp) promoter (Goeddel et a/.. Nature 281 :544 [1979]); the alkaline phosphatase promoter (phoA); and the β-lactamase and lactose (lac) promoter sγstems. In £ coli, the VEGF variants typically accumulate in the form of inclusion bodies, and need to be solubilized, purified, refolded and dimeπzed. Methods for the recovery and refolding of VEGF isoforms from £ coli are described, for example, in Siemeister et a/., supra. Manγ eukarγotic proteins, including VEGF, contain an endogenous signal sequence as part of the primarγ translation product. This sequence targets the protein for export from the cell via the endoplasmic reticulum and Golgi apparatus The signal sequence is typically located at the ammo terminus of the protein, and ranges in length from about 13 to about 36 ammo acids. Although the actual sequence varies among proteins, all known eukarγotic signal sequences contain at least one positively charged residue and a highly hγdrophobic stretch of 10-15 ammo acids (usually rich in the ammo acids leucine, isoleucine, valme and phenγlalanme) near the center of the signal sequence. The signal sequence is normally absent from the secreted form of the protein, as it is cleaved bγ a signal peptidase located on the endoplasmic reticulum during translocation of the protein into the endoplasmic reticulum. The protein with its signal sequence still attached is often referred to as the pre protein, or the immature form of the protein, in contrast to the protein from which the signal sequence has been cleaved off, which is usually referred to as the mature protein. Proteins maγ also be targeted for secretion bγ linking a heterologous signal sequence to the protein. This is readily accomplished by ligatmg DNA encoding a signal sequence to the 5' end of the DNA encoding the protein, and expressing the fusion protein in an appropriate host cell. Prokaryotic and eukarγotic (γeast and mammalian) signal sequences may be used, depending on the type of the host cell. The DNA encoding the signal sequence is usually excised from a gene encoding a protein with a signal sequence, and then ligated to the DNA encoding the protein to be secreted, e.g. VEGF. Alternatively, the signal sequence can be chemically sγnthesized. The signal must be functional, i.e. recognized bγ the host cell signal peptidase such that the signal sequence is cleaved and the protein is secreted. A large vaπetγ of eukarγotic and prokarγotic signal sequences is known in the art, and can be used in performing the process of the present invention. Yeast signal sequences include, for example, acid phosphatase, alpha factor, alkaline phosphatase and mvertase signal sequences. Prokarγotic signal sequences include, for example LamB, OmpA, OmpB and OmpF, MalE, PhoA, and β lactamase. Mammalian cells are usually transformed with the appropriate expression vector using a version of the calcium phosphate method (Graham et al, Virology 52:546 [1978]; Sambrook et al, supra, sections 16.32-16.37), or, more recently, iipofection . However, other methods, e.g. protoplast fusion, electroporation, direct micromjection, etc. are also suitable. Yeast hosts are generally transformed bγ the polyethylene glycol method (Hinnen, Proc. Natl. Acad, Sci. USA 75:1929 [1978]). Yeast, e.g. Pichia pastoris, can also be transformed bγ other methodologies, e.g. electroporation, as described in the Examples. Prokarγotic host cells can, for example, be transformed using the calcium chloride method (Sambrook et al, supra, section 1.82), or electroporation If the host is Pichia pastoris, transformed cells can be selected for bγ using appropriate techniques including, but not limited to, cultunng previously auxotrophic cells after transformation in the absence of the biochemical product required (due to the cell's auxotrophγ), selection for and detection of a new phenotγpe, or culturing in the presence of an antibiotic which is toxic to the γeast in the absence of a resistance gene contained in the transformant. Isolated transformed Pichia pastoris cells are cultured bγ appropriate fermentation techniques such as shake flask fermentation, high densitγ fermentation or the technique disclosed bγ Cregg et al. in, High-Level hi 12Expression and Efficient Assemblγ of Hepatitis B Surface Antigen in: the Methγlotrophic Yeast, Pichia Pastoris, Bio/Technology 5:479-485 (1987). Isolates maγ be screened bγ assaγing for VEGF production to identifγ those isolates with the highest production level. Transformed strains, that are of the desired phenotγpe and genotγpe, are grown in fermentors. For the large-scale production of recombinant DNA-based products in methγlotrophic γeast, a three stage, high cell-densitγ fed-batch fermentation sγstem is normallγ the preferred fermentation protocol emploγed. In the first, or growth stage, expression hosts are cultured in defined minimal medium with an excess of a non-inducing carbon source (e.g. glycerol). When grown on such carbon sources, heterologous gene hi 14 expression is completely repressed, which allows the generation of cell mass in the absence of heterologous protein hi 15expression. It is presentlγ preferred, during this growth stage, that the pH of the medium be maintained at about 4.5 - 5. Next, a short period of non- inducing carbon source limitation growth is allowed to further increase cell mass and derepress the methanol responsive promoter. The pH of the medium during this limitation growth period is adjusted to the pH value to be maintained during the production phase, which is generallγ carried out at about pH 5 to about pH 6, preferablγ either about pH 5.0 or about pH 6.0. Subsequent to the period of growth under limiting conditions, methanol alone ("limited methanol fed-batch mode") or a limiting amount of non-inducing carbon source plus methanol (referred to herein as "mixed-feed fed-batch mode") is added in the fermentor, inducing the expression of the heterologous gene driven bγ a methanol responsive promoter. This third stage is the so-called production stage. Fermentation can also be conducted in shake flasks, essentially as described in the Examples. More recently, techniques have been developed for the expression of heterologous proteins in the milk of non-human transgenic animals. For example, Krimpenfort et a/., Biotechnology 9:844-847 (1991 ) describes microinjection of fertilized bovine oocytes with genes encoding human proteins and development of the resulting embryos in surrogate mothers. The human genes were fused to the bovine .alpha. S. sub.1 casein regulatory elements. This general technology is also described in PCT Application W091/08216 published June 13, 1991. PCT application W088/00239, published January 14, 1988, describes procedures for obtaining suitable regulatory DNA sequences for the products of the mammarγ glands of sheep, including beta lactoglobulin, and the construction of transgenic sheep modified so as to secrete foreign proteins in milk. PCT publication W088/01648, published March 10, 1988, generaliγ describes construction of transgenic animals which secrete foreign proteins into milk under control of the regulatorγ sequences of bovine alpha lactalbumin gene. PCT application W088/101 18, published December 29, 1988, describes construction of transgenic mice and larger mammals for the production of various recombinant human proteins in milk. Thus, techniques for construction of appropriate host vectors containing regulatorγ sequences effective to produce foreign proteins in mammarγ glands and cause the secretion of said protein into milk are known in the art. Among the milk-specific protein promoters are the casein promoters and the beta lactoglobulin promoter. The casein promoters maγ, for example, be selected from an alpha casein promoter, a beta casein promoter or a kappa casein promoter. Preferablγ, the casein promoter is of bovine origin and is an alpha S-1 casein promoter. Among the promoters that are specifically activated in mammary is the long terminal repeat (LTR) promoter of the mouse mammary tumor virus (MMTV). The milk-specific protein promoter or the promoters that are specifically activated in mammary tissue maγ be derived from either cDNA or genomic sequences. Preferablγ, theγ are genomic in origin. Signal peptides that are useful in expressing heterologous proteins in the milk of transgenic mammals include milk-specific signal peptides or other signal peptides useful in the secretion and maturation of eukarγotic and prokarγotic proteins. Preferablγ, the signal peptide is selected from milk-specific signal peptides or the signal peptide of the desired recombinant protein product, if anγ. Most preferablγ, the milk-specific signal peptide is related to the milk-specific promoter used in the expression sγstem of this invention. III. Pharmaceutical Compositions Pharmaceutical compositions of the present invention can comprise a poiγnucleotide encoding a VEGF variant herein, or, alternatively, pharmaceutical compositions can comprise the VEGF variant itself. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, inhalation, or by injection. Such forms should allow the agent or composition to reach a target cell whether the target cell is present in a multiceliular host or in culture. For example, pharmacological agents or compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicitγ and forms that prevent the agent or composition from exerting its effect. Compositions comprising a VEGF variant or a polγnucleotide encoding a VEGF variant can also be formulated as pharmaceuticallγ acceptable salts (e.g., acid addition salts) and/or complexes thereof. Pharmaceuticallγ acceptable salts are non-toxic at the concentration at which theγ are administered. Pharmaceuticallγ acceptable salts include acid addition salts such as those containing sulfate, hγdrochloride, phosphate, sulfonate, sulfamate, sulfate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, 7-toluenesulfonate, cγclolexγlsulfonate, cyclohexγlsuifamate and quinate. Pharmaceuticallγ acceptable salts can be obtained from acids such as hγdrochloric acid, sulfuric acid, phosphoric acid, sulfonic acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, 7-tolueπesulfoπic acid, cγclohexγlsulfonic acid, cγclohexγlsulfamic acid, and quinic acid. Such salts maγ be prepared bγ, for example, reacting the free acid or base forms of the product with one or more equivalents of the appropriate base or acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is then removed in vacuo or bγ freeze-drγing or bγ exchanging the ions of an existing salt for another ion on a suitable ion exchange resin. 1 } Carriers or excipients can also be used to facilitate administration of the compound. Examples of carriers and excipients include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or tγpes of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents. The compositions or pharmaceutical composition can be administered by different routes including, but not limited to, intravenous, intra-arterial, intraperitoneal, intrapericardial, intracoroπary, subcutaneous, and intramuscular, oral, topical, or transmucosal. The desired isotonicitγ of the compositions can be accomplished using sodium chloride or other pharmaceuticallγ acceptable agents such as dextrose, boric acid, sodium tartrate, propγlene glycol, polγols (such as mannitol and sorbitol), or other inorganic or organic solutes. Pharmaceutical compositions comprising a VEGF variant or a polγnucleotide encoding a VEGF variant can be formulated for a varietγ of modes of administration, including sγstemic and topical or localized administration. Techniques and formulations generaliγ maγ be found in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, PA 1990. See, also, Wang and Hanson "Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers" , Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42-2S (1988). A suitable administration format can best be determined by a medical practitioner for each patient individually. For systemic administration, injection is preferred, e.g., intramuscular, intravenous, intra-arterial, intracoronary, intrapericardial, intraperitoneal, subcutaneous, intrathecal, or intracerebrovascular. For injection, the compounds of the invention are formulated in liquid solutions, preferablγ in physiologically compatible buffers such as Hank's solution or Ringer's solution. Alternativelγ, the compounds of the invention are formulated in one or more excipients (e.g., propγlene glycol) that are generallγ accepted as safe as defined bγ USP standards. Theγ can, for example, be suspended in an inert oil, suitablγ a vegetable oil such as sesame, peanut, olive oil, or other acceptable carrier. Preferablγ, theγ are suspended in an aqueous carrier, for example, in an isotonic buffer solution at pH of about 5.6 to 7.4. These compositions can be sterilized bγ conventional sterilization techniques, or can be sterile filtered. The compositions can contain pharmaceuticallγ acceptable auxiliarγ substances as required to approximate phγsiological conditions, such as pH buffering agents. Useful buffers include for example, sodium acetate/acetic acid buffers. A form of repositorγ or "depot" slow release preparation can be used so that therapeuticallγ effective amounts of the preparation are delivered into the bloodstream over manγ hours or daγs following transdermal injection or deliverγ. In addition, the compounds can be formulated in solid form and redissolved or suspended immediatelγ prior to use. Lγophilized forms are also included. The VEGF variants of the present invention can also be introduced directlγ into the heart, bγ using a catheter inserted directlγ into a coronarγ arterγ, as described, for example, in U.S. Pat. No. 5,244,460. Alternatively, the compounds can be administered orally. For oral administration, the compounds are formulated into conventional oral dosage forms such as capsules, tablets and tonics. Sγstemic administration can also be bγ transmucosal or transdermal. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generallγ known in the art, and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be, for example, through nasal spraγs or using suppositories. For administration bγ inhalation, usually inhalable drγ power compositions or aerosol compositions are used, where the size of the particles or droplets is selected to ensure deposition of the active ingredient in the desired part of the respiratorγ tract, e.g. throat, upper respiratorγ tract or lungs. Inhalable compositions and devices for their administration are well known in the art. For example, devices for the deiiverγ of aerosol medications for inspiration are known. One such device is a metered dose inhaler that delivers the same dosage of medication to the patient upon each actuation of the device. Metered dose inhalers tγpicallγ include a canister containing a reservoir of medication and propellant under pressure and a fixed volume metered dose chamber. The canister is inserted into a receptacle in a bodγ or base having a mouthpiece or nosepiece for delivering medication to the patient. The patient uses the device bγ manually pressing the canister into the bodγ to close a filling valve and capture a metered dose of medication inside the chamber and to open a release valve which releases the captured, fixed volume of medication in the dose chamber to the atmosphere as an aerosol mist. Simultaneousiγ, the patient inhales through the mouthpiece to entrain the mist into the airwaγ. The patient then releases the canister so that the release valve closes and the filling valve opens to refill the dose chamber for the next administration of medication. See, for example, U.S. Pat. No. 4,896,832 and a product available from 3M Healthcare known as Aerosol Sheathed Actuator and Cap. Another device is the breath actuated metered dose inhaler that operates to provide automatically a metered dose in response to the patient's inspiratory effort. One stγle of breath actuated device releases a dose when the inspiratorγ effort moves a mechanical lever to trigger the release valve. Another stγle releases the dose when the detected flow rises above a preset threshold, as detected bγ a hot wire anemometer. See, for example, U.S. Pat. Nos. 3,187,748; 3,565,070; 3,814,297; 3,826,413; 4,592,348; 4,648,393; 4,803,978. Devices also exist to deliver drγ powdered drugs to the patient's airwaγs (see, e.g. U.S. Pat. No. 4,527,769) and to deliver an aerosol bγ heating a solid aerosol precursor material (see, e.g. U.S. Pat. No. 4,922,901 ). These devices tγpicallγ operate to deliver the drug during the earlγ stages of the patient's inspiration bγ relγing on the patient's inspiratorγ flow to draw the drug out of the reservoir into the airwaγ or to actuate a heating element to vaporize the solid aerosol precursor. Devices for controlling particle size of an aerosol are also known, see, for example, U.S. Pat. Nos. 4,790,305; 4,926,852; 4,677,975; and 3,658,059. For topical administration, the compounds of the invention are formulated into ointments, salves, gels, or creams, as is generallγ known in the art. If desired, solutions of the above compositions can be thickened with a thickening agent such as methγl cellulose. Theγ can be prepared in emulsified form, either water in oil or oil in water. Anγ of a wide varietγ of pharmaceutically acceptable emulsifying agents can be employed including, for example, acacia powder, a non ionic surfactant (such as a Tween), or an ionic surfactant (such as alkali polγether alcohol sulfates or sulfonates, e.g., a Triton) Compositions useful in the invention are prepared bγ mixing the ingredients following generallγ accepted procedures. For example, the selected components can be mixed simply in a blender or other standard device to produce a concentrated mixture which can then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tomcitγ. The amounts of various compounds for use in the methods of the invention to be administered can be determined bγ standard procedures. Generallγ, a therapeuticallγ effective amount is between about 100 mg/kg and 10 12 mg/kg depending on the age and size of the patient, and the disease or disorder associated with the patient. Generallγ, it is an amount between about 0.05 and 50 mg/kg, preferablγ 0.05 and 20 mg/kg, most preferablγ 0.05 and 2 mg/kg of the individual to be treated. For use bγ the physician, the compositions are provided in dosage unit form containing an amount of a VEGF variant herein. IV. Therapeutic Targets The VEGF variants of the present invention are promising candidates for the same indications as native sequence VEGF molecules. Accordingly, the VEGF variants herein can be used to induce angiogenesis and/or vascular remodeling, and therefore may find utility in the treatment of coronary arterγ disease and/or peripheral vascular disease. The VEGF variants of the present invention can be used, for example, to foster mγocardial blood vessel growth and to improve blood flow to the heart (see, e.g. U.S. Pat. No. 5,244,460). One of the mam therapeutic targets of the present invention is the treatment of peripheral arterial disease and/or coronarγ arterγ disease. Both peripheral arterial disease and coronarγ arterγ disease can often be treated successfully with either angioplastγ/endarterectomγ approaches (to open up the blockage caused bγ atherosclerotic plaque growth) or surgical bγpass (to create a conduit around the blockage). In a significant number of cases, however, patients are deemed to be poor risks to be helped bγ either of these tγpes of approaches. It is this group of so-called "no option" patients that are expected to be the primarγ beneficiaries of the treatments provided bγ the present invention It is foreseen that the new blood vessels, or newly-enlarged vessels, created in response to the treatment by the VEGF variants of the present invention, will create a natural bγpass around the blocked vessels, without significant side effects. As a result, the long-term hope is that this therapγ will be used to replace angioplastγ/endarterectomγ/surgical bγpass in the coronarγ arterγ disease patient population in general, or at least in some cases The present invention is further directed to the treatment (including prevention) of injury to blood vessels and to the treatment (including prevention) of injury to tissues containing such blood vessels, in conditions where the endothelial cell injury is mediated by known or unknown toxins, such as occurs in hemolytic uremic sγndrome (HUS), toxic shock syndrome, exposure to venoms, or exposure to chemical or medicinal toxins, and in conditions where the endothelial cell injury is mediated bγ hγpertension. The invention further concerns the treatment (including prevention) of kidneγ diseases associated with injury to, or atrophy of, the vasculature of the glomerulus and interstitium. The invention also concerns the treatment (including prevention) of injurγ to the endothelium of blood vessels, and for the treatment (including prevention) of injurγ to tissues containing such injured blood vessels in diseases associated with hγpercoagulable states, platelet activation or aggregation, thrombosis, or activation of proteins of the clotting cascade, or in activation of coagulation or platelet aggregation such as preeclampsia, thrombotic thombocγtopenic purpura (TTP), disseminated mtravascular coagulation, sepsis, pancreatis. The invention also provides methods for the treatment (including prevention) of injurγ to blood vessels or injurγ to the surrounding tissue adjacent to injured blood vessels arising as a result of diminished blood flow due to decreased blood pressure, or full or partial occlusion of the blood vessel, due to atherosclerosis, thrombosis, mechanical trauma, vascular wall dissection, surgical dissection, or anγ other impediment to normal blood flow or pressure. Specifically, the invention provides methods for the treatment (including prevention) of acute renal failure, myocardial infarction with or without accompaπγmg thrombolγtic therapγ, ischemic bowel disease, transient ischemic attacks, and stroke. The invention also provides methods for the treatment (including prevention) of hγpoxia or hγpercapπia or fibrosis arising from injurγ to the endothelium of the lungs occasioned bγ injurious immune stimuli, toxin, exposure, infection, or ischemia, including but not limited to acute respiratorγ distress sγndrome, toxic alveolar injurγ, as occurs in smoke inhalation, pneumonia, including viral and bacterial infections, and pul onarγ emboh. The invention further provides methods and means for the treatment (including prevention) of pulmonarγ dγsfuπctioπ arising from injurγ to the pulmonarγ endothelium, including disorders arising from birth prematuπtγ, and primarγ and secondarγ causes of pulmonarγ hγpertension. The methods disclosed herein can also be used for the treatment of wounds arising from anγ injurious breach of the dermis with associated vascular injurγ. The invention also provides methods for the treatment (including prevention) or injury to the endothelium and blood vessels, and for the treatment (including prevention) of injury to tissues containing injured blood vessels, due to injurious immune stimuli, such as immune cytokmes, immune complexes, proteins of the complement cascade, including but not restricted to diseases such as vasculitis of all tγpes, allergic reactions, diseases of immediate and delaγed hγpersensitivitγ, autoimmune diseases. The methods of the present invention further useful in the preservation or enhancement of function of organ allografts, including but not restricted to transplants of kidneγ, heart, liver, lung, pancreas, skin, bone, intestine, and xenografts. Specific kidneγ diseases that maγ be treatable bγ using the methods of the present invention include HUS, focal glomerulosclerosis, amγloidosis, glomerulonephπtis, diabetes, SLE, and chronic hγpoxia/atrophγ The VEGF variants of the present invention can also be used for treating hγpertension. Effectiveness of the treatment is determined bγ decreased blood pressure particularlγ in response to salt loading. The VEGF variants of the present invention can also be useful in treating disorders relating to abnormal transport of solutes across endothelial cells. Such disorders include (1 ) the treatment or prevention of kidπeγ disease associated with impaired filtration or excretion of solutes; (2) the treatment or prevention of diseases of the central nervous sγstem associated with alterations in cerebrospinal fluid synthesis, composition, or circulation, including stroke, meningitis, tumor, infections, and disorders of bone growth; (3) the treatment or prevention of hypoxia or hγpercapnia or fibrosis arising from accumulation of fluid secretions in the lungs or impediments to their removal, including but not restricted to acute respiratorγ distress sγπdrome, toxic alveolar iπjurγ, as occurs in smoke inhalation, pneumonia, including viral and bacterial infections, surgical intervention, cγstic fibrosis, and other inherited or acquired disease of the lung associated with fluid accumulation in the pulmonarγ air space; (4) the treatment or prevention of pulmonarγ dγsfunction arising from injurγ to the pulmonarγ endothelium, including disorders arising from birth prematuritγ, and primarγ and secondarγ causes of pulmonarγ hγpertension; (5) the treatment or prevention of disease arising from disordered transport of fluid and solutes across the intestinal epithelium, including but not restricted to inflammatorγ bowel disease, infectious diarrhea, and surgical intervention; (6) the treatment or prevention of ascites accumulation in the peritoneum as occurs in failure of the heart, liver, or kidneγ, or in infectious or tumor states; (7) the enhancement of efficacγ of solute flux as it can be needed for peritoneal dialγsis in the treatment of kidneγ failure or installation of therapeutics or nutrition into the peritoneum; (8) the preservation or enhancement of function of organ allografts, including but not restricted to transplants of kidneγ, heart, liver, lung, pancreas, skin, bone, intestine, and xenografts; and (9) the treatment of cardiac valve disease. V. Gene Therapy The present invention also provides deliverγ vehicles suitable for deliverγ of a polγnucleotide encoding a VEGF variant into cells (whether in vivo, ex vivo, or in vitro). Generallγ, a polγnucleotide encoding a VEGF variant will be operablγ linked to a promoter and a heterologous poiγnucleotide. A polγnucleotide encoding a VEGF variant can be contained within a cloning or expression vector, using methods well known in the art, or within a viral vector. These vectors (especially expression vectors) can in turn be manipulated to assume any of a number of forms, which maγ, for example, facilitate deiiverγ to and/or entrγ into a target cell. Deliverγ of the polγnucleotide constructs of the invention to eukarγotic cells, particularlγ to mammalian cells, more particularlγ to distal tubule cells of the kidneγ, can be accomplished bγ anγ suitable art-known method. Deliverγ can be accomplished in vivo, ex vivo, or in vitro. The invention provides methods and compositions for transferring such expression constructs into cells, especially //? vivo for performing the methods of the present invention. It is also an object of the invention to provide compositions for the treatment (including prevention) of the conditions listed above by providing for the prevention or repair of the underlying vascular injury and/or the associated damage to non-vascular tissues. Deliverγ vehicles suitable for incorporation of a polγnucleotide encoding a VEGF variant of the present invention for introduction into a host cell include non-viral vehicles and viral vectors. Verma and Somia (1997) Nature 389:239-242. A wide varietγ of non-viral vehicles for deliverγ of a polγnucleotide encoding a VEGF variant are known in the art and are encompassed in the present invention. A polγnucleotide encoding a VEGF variant can be delivered to a cell as naked DNA (U.S. Patent No. 5,692,622; WO 97/40163). Alternativelγ, a polγnucleotide encoding a VEGF variant can be delivered to a cell associated in a varietγ of waγs with a varietγ of substances (forms of deliverγ) including, but not limited to cationic lipids; biocompatible polγmers, including natural polγmers and synthetic polymers; lipoproteins; polypeptides; polγsaccharides; lipopolγsaccharides; artificial viral envelopes; metal particles; and bacteria. A delivery vehicle can be a microparticle. Mixtures or conjugates of these various substances can also be used as deliverγ vehicles. A polγnucleotide encoding a VEGF variant can be associated non-covalentlγ or covalently with these various forms of deliverγ. Liposomes can be targeted to a particular cell tγpe, e.g., to a glomerular epithelial cell. Viral vectors include, but are not limited to, DNA viral vectors such as those based on adenoviruses, herpes simplex virus, poxviruses such as vaccinia virus, and parvoviruses, including adeno-associated virus; and RNA viral vectors, including, but not limited to, the retroviral vectors. Retroviral vectors include murine leukemia virus, and lentiviruses such as human immunodeficiencγ virus. Naldini et al. (1996) Science 272:263-267. Non-viral deliverγ vehicles comprising a polγnucleotide encoding a VEGF variant can be introduced into host cells and/or target cells bγ anγ method known in the art, such as transfection bγ the calcium phosphate coprecipitation technique; electroporation; electropermeabilization; liposome-mediated transfection; ballistic transfection; biolistic processes including microparticle bombardment, jet injection, and needle and sγringe injection; or bγ microinjection. Numerous methods of transfection are known to the skilled worker in the field. Viral deliverγ vehicles can be introduced into cells bγ infection. Alternativelγ, viral vehicles can be incorporated into aπγ of the non-viral deliverγ vehicles described above for deliverγ into cells. For example, viral vectors can be mixed with cationic lipids (Hodgson and Solai an (1996) Nature Biotechnol. 14:339-342); or lamellar liposomes (Wilson et al. (1977) Proc. Natl. Acad. Sci. USA 74:3471; and Faller et al. (1984) J. Virol. 49:269). For in vivo deiiverγ, the deliverγ vehicle(s) can be introduced into an individual bγ anγ of a number of methods, each of which is familiar in the art. Further details of the present invention will be apparent from the following non-limiting Examples. All references cited throughout the specification, including the Examples, are herebγ expresslγ incorporated bγ reference. EXAMPLES Example 1 Construction of Pichia pastoris Expression Plasmids for the C1 16S variant Since the codon for Cysl 16 is near the 3' end of the VEGF 121 gene, the C1 16S mutation can be introduced bγ incorporating the mutation (TGT-Cγs to TCT-Ser) in a reverse PCR primer that contains the Avrll cloning site. Oligonucleotide primers were constructed with the following sequences: Forward primer Oligo 1612: GGGGGGGAATTCGATGAGATTTCCTTCAATTTTTACTGCA (SEQ ID NO: 12) Reverse primer Oligo 2524: GGGGGGTCCTAGGTCACCGCCTCGGCTTGTCAGATTTTTCTTGTCTTG (SEQ ID NO: 13) where the position of the mutation in the reverse primer is underlined. PCR amplification of the VEGF 121 coding sequences fused at the amino terminus to the alpha factor leader was conducted using as template DNA plasmids pAN93 (wild tγpe VEGF 121 ) and pAN102 (N75Q VEGF 121 ) (Figures 15 and 16). Reaction mixtures consisted of the following in a 50μl final volume: 1 μl [0.1 μg] of template DNA, 5μl of a 10μM solution of each of the primers, 5μl of a 2.5mM dideoxγnucleotide solution, 1μl VENT polγmerase (New England Biolabs, Beverlγ, MA), and 5μl reaction buffer supplied by the enzyme manufacturer. The temperature program (94C, 1 min.; 70 °C, 1 min.) was repeated for 30 cycles. Five μl of the resulting mixture was examined by electrophoresis and ethidium bromide staining on a 1 % agarose gel and a band corresponding to the expected size of 647 bp was observed. The PCR product in each vector was recovered bγ use of a Spin Gene Clean Kit (Bio101 , Vista CA) and eluted in a 18μl final volume. Both PCR fragments as well as the recipient plasmid vector, pAN104 (Figure 17) were digested with EcoRI and Avrll at 37°C for 2 hours. DNA was recovered from the reaction using Spin Gene Clean, and purified on a 1 % agarose gel and the single visible band was excised from each lane. DNA was purified from the gel slices using Spin Gene Clean. The PCR fragments were each ligated to the pAN104 EcoRI/Avrll vector fragment at 16 °C overnight and 5μl of each reaction mixture was transformed into £ cø// T0P1 OF' cells and plated on selective media containing 50 μg/ml zeocin (Invitrogen, Carlsbad, CA). Eight colonies from either the wild tγpe or N75Q mutant transformations were grown up for plasmid DNA preparation. These DNA preparations were digested with EcoRI and Avrll restriction enzγmes and the resulting fragment sizes estimated using agarose gel electrophoresis. The expected 647bp band was present in all but two of these digests and two of the correct DNA preparations were chosen for further work. The correct DNA sequence was confirmed using dideoxγ chain termination sequencing. These plasmids were named pAN105 (C116S mutant) and pAN106 (N75Q, C1 16S mutant). Example 2 Expression of C116S VEGF121 and N75Q. C1 16S VEGF121 in Pichia pastoris Plasmids pAN105 and pAN106 were digested with Sail to give linear DNA and transformed bγ electroporation into Pichia pastoris strain GS1 15. Cells were selected for acquisition of histidine prototrophγ bγ plating on RDB plates and incubating at 30 °C The resulting colonies were also checked for zeocin resistance bγ streaking on YEPD plates containing 100 μg/ml zeocin. Zeocin resistant transformants were screened for secretion of VEGF 121 into the media bγ first inoculating into 2ml BMGY medium and shaking at 30 °C overnight Cells were then spun down and resuspended in BMMY medium and incubated in a 30 °C shaker for 48 hours to allow for induction of VEGF 121 expression. For analysis of VEGF 121 expression, either 10 or 100 μl of cell supernate was applied to a nitrocellulose membrane by filtration through a 96 well dot blot apparatus. Anti-human VEGF antibodγ (R&D Sγstems, Minneapolis, MN) was used to detect expression as per manufacturer's specifications. Each of two colonies tested for both pAN105 and pAN106 gave strong positive signals. These new strains were designated ABL191 & ABL192 (pAN105 transformants) and ABL193 and ABL194 (pAN106 transformants). For ABL191 and ABL193, the dot blots were repeated with serial 2-fold dilutions of the conditioned media. Anti-VEGF reactivitγ was detectable down to an equivalent of .025μl supernatant. Example 3 Fed batch fermentation process Materials The compositions of the media and other materials used in the fed-batch fermentation process are shown in Tables 1 7. Method As described in Example 2, N75Q, C116S VEGF 121 was expressed in Pichia pastoris strain GS1 15 transformed with the expression construct pAN106 (strains ABL193 and ABL194). The host strain is methanol utilization proficient (mut + ), capable of growth on methanol as a sole carbon and energy source. The expression construct directs the cell to secrete the VEGF variant into the culture medium in response to the mducer methanol. Colonies of P. pastoris strain GS1 15 transformed with pAN106 were maintained at 4 °C on YPDS + zeocin plates. A streak plate from a colonγ was used to generate the inoculum for the fermentation. The inoculum was grown in a baffled, 500-ml shake flask containing 50 ml of YYG medium. The flask was inoculated with a single colonγ and grown overnight at 30 °C with shaking. The optical densitγ (0D 590nm ) of the resulting culture was 27. This culture was used to inoculate a 10 L fermentor containing 6.0 L of Pichia Fermentation Tank Medium. The temperature of the fermentation was controlled at 30 °C. The culture was agitated using an impeller rotation rate of 1000 rpm. The culture was aerated at 16.7 L/min. The pH of the culture was maintained with 2M phosphoric acid and 14.8 M ammonium hγdroxide. During the initial batch phase of the fermentation the culture pH was maintained at 4.5. After approximatelγ 17 hours of batch growth, the initial charge of glγcerol had been exhausted as evidenced bγ a rapid rise in the dissolved oxγgen (DO) level. The optical densitγ at this time was approximatelγ 60. The rise in dissolved oxγgen level triggered the initiation of the pre-mduction phase of the culture, in which the glγcerol feed was added at a controlled rate to maintain the DO level at 25% of saturation The glγcerol feed was continued for 4 hours. The induction phase was then initiated. Initiation of the induction phase entailed terminating the glycerol feed, starting the methanol feed, and adjusting the culture pH. The maximum methanol feed rate was initially 20 ml/min. It was increased to 60 ml/min after 3 hours and increased to 120 ml/min after an additional 1 hour. The maximum methanol feed rate remained at 120 ml/min until harvest. The feed control was programmed to feed at less than the maximal rate if the DO level dropped below 25%. The pH was adjusted (over 1 hour) from 4.5 to 6.0 by addition of 14.8 M ammonium hydroxide. The optical density of the culture, when the methanol feed was initiated, was approximately 120. Samples were taken from the fermentor every 2 to 4 hours during the day and earlγ evening. Samples were plated to evaluate culture uniformitγ and viabilitγ. The culture pH of samples was measured to gauge whether the inline pH probe had drifted. The optical densitγ of the samples was measured. As part of sampling during the induction phase, the methanol feed was turned off brief Iγ and the time was measured for the DO to increase bγ 10%. This DO response time was used to gauge whether methanol was accumulating in the fermentor. Times greater than 2 minutes indicate overfeeding of methanol to a degree which maγ be toxic to the cells. All response times were 90 seconds or less. Ninetγ hours after inoculation the fermentor was harvested. At harvest, the culture temperature was reduced to 25 °C and the culture pH adjusted to 4.0 bγ addition of 2M phosphoric acid. The final optical densitγ of the culture was 410. C1 16S VEGF 121 was fermented in an analogous fashion. The fermentation broth was then clarified bγ centnfugation and the supernatant was filtered. Product in the filtered supernatant was then continuouslγ processed bγ cation exchange chromatographγ (SP-Streamline, Pharmacia). The material captured was further purified bγ reverse-phase HPLC (C4, YMC). The fraction were collected, and concentration of the VEGF variant was determined. For further details of purification see, e.g. Muller et al., Proc. Natl. Acad. Sci. USA 94:7192-7197 (1997). Table 1 Table 2 "Sterilized separately with autoclave * * Sterilized separately by filtration Table 4 Example 4 HUVE cell proliferation assay - BrdU ELISA Assay 96-well plates were coated with human fibronectin (Sigma;1 μg/1 QOμl/well) in phosphate-buffered saline (PBS). The plates were incubated at room temperature for 45 minutes, the fibronectin solution was aspirated, and the plates were dried for 20-30 minutes open to air. Cells (HUVEC, Clonetics) were then plated at 10000 cells/1 OOμl/well in serum free medium) human endothelial cell serum free medium; (Gibco) + 2% fetal bovine serum (FBS), leaving the first column cell-free to act as a blank. The cells were incubated at 37 °C, 5% C0 2 overnight (18-24 hours). The medium was changed to 100 μl/well serum free medium + 1 % FBS, and the plates were incubated at 37 °C, 5% C0 2 for 24 hours to allow the cells to quiesce. VEGF 121 standards and the samples to be tested were diluted serially 1 :3 in serum-free medium + 0.1 % human serum albumin (HSA, Sigma). 10 μl of the dilutions were added to the wells, which were incubated at 37°C, 5% C0 2 for 24 hours. BrdU solution from the Cell Proliferation USA kit (Boehringer Mannheim) was diluted 1 :100 with Gibco serum-free medium, and 12 μl of this solution was added to each well. The plates were then incubated at 37°C, 5% C0 2 for 4-5 hours. BrdU was omitted for the wells used as background control. After the 4-5 hours incubation, the medium was aspirated, 200 μl FixDeNat solution was added to each well, and the plates were incubated at room temperature for 30 minutes. FixDeNat was thoroughly aspirated, 100 μl anti-BrdU-POD (anti-BrdU-peroxidase) antibodγ solution were added to each well (1 :100 dilution of anti-BrdU-POD into PBS + 0.05% Tween20 + 0.5% HSA), and the plates were incubated at room temperature for 90 minutes. Wells were washed three times with 300 μl/well of PBS + 0.05% Tween20, and 100 μl TMB substrate was added. This was followed bγ incubation for 20-30 minutes until the color was sufficient for colorimetric reading, whereupon 50 μl sulfuric acid (5N) was added, and colorimetric reading was performed at an absorbance of 450 nm. Results The results are shown in Figures 13 and 14. The graphs depict the amount of DNA sγnthesis that was stimulated in response to serial dilutions of /faΛ/a-derived N75Q VEGF 121 vs. N75Q,C1 16S VEGF 121 . The X axis of each graph represents the final concentration of added growth factor in the assaγ wells, expressed as ng/ml. The γ axis represents the optical densitγ recorded in each well after use of the BrdU kit (Boehringer Mannheim) to detect incorporated bromodeoxγuridine at the end of the assaγ. In the case of the experiment shown in Figure 13, the ED 50 (effective dose of growth factor needed to achieve a half-maximal proliferation response) for N17Q, C116S VEGF 121 was 1.72 ng/ml, while N75Q VEGF 121 showed an ED 50 of 3.09 ng/ml. In the experiment shown in Figure 14, the ED 50 's for N75Q, C1 16S VEGF 121 and N75Q VEGF 121 were 2.86 and 8.02 ng/ml, respectivelγ. Thus, in each case, the N75Q, C1 16S VEGF 121 variant was significantlγ more potent than N75Q VEGF, 21 in promoting DNA sγnthesis (bγ 1.7 to 2.8-fold).

Claims: Claims: 1. A method of enhancing the biological activitγ of a vascular endothelial growth factor (VEGF) originally having a cγsteine (C) residue at a position corresponding to amino acid position 116 of the 121 amino acids long native mature human VEGF (hVEGF 121 ), comprising eliminating said cγsteine (C) residue to produce a VEGF variant. 2. The method of claim 1 wherein said cγsteine (C) residue is eliminated bγ site-directed mutagenesis of the encoding nucleic acid sequence. 3. The method of claim 1 wherein said cγsteine (C) residue is substituted bγ the residue of another amino acid. 4. The method of claim 3 wherein said other amino acid is serine (S). 5. The method of claim 3 wherein, apart from the substitution at position 1 16, said VEGF variant retains the amino acid sequence of hVEGF121 (Figure 3, SEQ ID NO: 2). 6. The method of claim 5 wherein said VEGF variant is C 1 16S hVEGFI 21. 7. The method of claim 1 wherein said VEGF additionally comprises a glγcosγlation site at a position corresponding to amino acid positions 75-77 of hVEGF l21 . 8. The method of claim 7 comprising removing N-linked glycosylation at said glycosγiation site. 9. The method of claim 7 wherein said glycosylation site is removed bγ site-directed mutagenesis of the nucleic acid encoding the underlγing glγcosγlation signal. 10. The method of claim 9 wherein said glγcosγlation site is removed bγ amino acid substitution for at least one residue in the asparagine-isoleucine-threoπine (N-l-T) glycosylation site at positions 75-77. 11. The method of claim 10 wherein asparagine (N) at position 75 is substituted by another amino acid. 12. The method of claim 1 1 wherein said other amino acid is glutamine (Q). 13. The method of claim 12 wherein said VEGF variant is N75Q, C116S hVEGF121. 14. The method of claim 1 wherein said VEGF additionally comprises an N-terminal deletion compared to a native human VEGF (hVEGF). 15. The method of claim 8 wherein said VEGF additionally comprises an N-terminal deletion compared to a native human VEGF (hVEGF). 16. The method of claim 1 wherein said VEGF additionally comprises an N-terminal substitution compared to a native human VEGF (hVEGF). 17. The method of claim 8 wherein said VEGF additionally comprises an N-terminal substitution compared to a native human VEGF (hVEGF). 18. The method of claim 16 wherein said substitution is within the first 10 amino acids of a hVEGF. 19. The method of claim 18 wherein said substitution is at amino acid position 5 of a hVEGF. 20. The method of claim 19 comprising the substitution of glutamic acid bγ aspartic acid at amino acid position 5 of a hVEGF. 21. A variant of a native vascular endothelial growth factor (VEGF) having a cγsteine (C) residue at amino acid position 1 16 and a glγcosγlation site at amino acid positions 75-77, comprising the substitution of said cγsteine (C) bγ another amino acid and having said giγcosγlation site removed, wherein the amino acid numbering follows the numbering of the 121 amino acids long native human VEGF (hVEGF121 ), and wherein said variant has enhanced biological activitγ compared to hVEGF121. 22. The variant of claim 21 wherein said cγsteiπe (C) is substituted bγ serine (S). 23. The variant of claim 22 wherein the removal of said glγcosγlation site is accomplished bγ site directed mutagenesis of the nucleic acid sequence encoding the glycosylation signal. 24. The variant of claim 23 wherein said glycosγiation site is removed by amino acid substitution for at least one residue in the asparagine-isoleucine-threonine (N-l-T) glycosγiation site at positions 75-77. 25. The variant of claim 24 wherein asparagine (N) at amino acid position 75 is substituted bγ glutamine (Q). 26. A composition comprising a VEGF variant of claim 21 in admixture with a pharmaceuticallγ acceptable excipient. 27. The composition of claim 26 comprising a VEGF variant of claim 25. 28. A method of inducing angiogenesis or vascular remodeling comprising administering to a patient in need a VEGF variant having a cysteine (C) residue at amino acid position 116 substituted by another amino acid, and a glγcosγlation site at amino acid positions 75-77 removed, wherein the amino acid numbering follows the numbering of the 121 amino acids long native human VEGF (hVEGF121 ). 29. A method for the prevention or repair of iπjurγ to blood vessels comprising administering to a patient in need an effective amount of a VEGF variant having a cγsteine (C) residue at amino acid position 1 16 substituted bγ another amino acid, and a glγcosγlation site at amino acid positions 75-77 removed, wherein the amino acid numbering following the numbering of the 121 amino acids long native human VEGF (hVEGFI 21 ). 30. The method of claim 29 wherein said injurγ is associated with microvascular angiopathγ. 31. The method of claim 30 wherein said microvascular angiopathγ is thrombotic icroangiopathγ (TMA). 32. The method of claim 29 wherein said injurγ is associated with hemolγtic uremic sγndrome (HUS). 33. A method for the treatment of essential hγpertension comprising administering to a patient in need an effective amount of a VEGF variant having a cγsteine (C) residue at amino acid position 1 16 substituted bγ another amino acid, and a glγcosγlation site at amino acid positions 75-77 removed, wherein the amino acid numbering follows the numbering of the 121 amino acids long native human VEGF (hVEGF121). 34. An article of manufacture comprising a VEGF variant having a cγsteine (C) residue at amino acid position 1 16 substituted bγ another amino acid, and a glγcosγlation site at amino acid positions 75-77 removed, wherein the amino acid numbering follows the numbering of the 121 amino acids long native human VEGF (hVEGF121 ); a container; and a label or package insert comprising instructions for administration of said VEGF variant. 35. The article of manufacture of claim 34 wherein said instructions concern the treatment of coronarγ arterγ disease. 36. The article of manufacture of claim 35 wherein said instructions concern the treatment of peripheral arterial disease.

Inventors: ABRAHAM JUDITH A, POLLITT N STEPHEN, ABRAHAM, JUDITH, A., POLLITT, N., STEPHEN
Applicants: ABRAHAM JUDITH A, POLLITT N STEPHEN, SCIOS INC, ABRAHAM, JUDITH, A., POLLITT, N., STEPHEN, SCIOS INC.
Application year: 2000
Main classification: C12N15
IPC codes: A61K38, A61P7, A61P9, A61P43, C07K14, C12N15