Steven,
The best will be that you again call HGSI and ask for clarification on VEGF-1/2??
ac = amino acid
ec = endothelial cell
Miljenko
For input on VEGF isoforms and new VEGF type, VEGF-B, read this part from patent:
United States Patent 5,840,693
Eriksson , et al. November 24, 1998
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Vascular endothelial growth factor-B
Abstract
VEGF-B polypeptides from the PDGF family of growth factors having the property of promoting mitosis and proliferation of vascular endothelial cells, DNA sequences encoding these polypeptides, pharmaceutical compositions containing them and antibodies which react with them. The VEGF-B polypeptides are useful in stimulating angiogenesis as well as in diagnostic applications.
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Inventors: Eriksson; Ulf (B.ang.lsta, SE); Olofsson; Birgitta (Stockholm, SE); Alitalo; Kari (Espoo, FI); Pajusola; Katri (Helsinki, FI)
Assignee: Ludwig Institute for Cancer Research (New York, NY); Helsinki University Licensing Ltd. Oy (Helsinki, FI)
Appl. No.: 609443
Filed: March 1, 1996
BACKGROUND OF THE INVENTION
Angiogenesis, or the proliferation of new capillaries from pre-existing blood vessels, is a fundamental process necessary for normal growth and development of tissues. It is a prerequisite for the development and differentiation of the vascular tree, as well as for a wide variety of fundamental physiological processes including embryogenesis, somatic growth, tissue and organ repair and regeneration, cyclical growth of the corpus luteum and endometrium, and development and differentiation of the nervous system. In the female reproductive system, angiogenesis occurs in the follicle during its development, in the corpus luteum following ovulation and in the placenta to establish and maintain pregnancy. Angiogenesis additionally occurs as part of the body's repair processes, e.g. in the healing of wounds and fractures. Angiogenesis is also a factor in tumor growth, since a tumor must continuously stimulate growth of new capillary blood vessels in order to grow.
Capillary blood vessels consist of endothelial cells and pericytes. These two cell types carry all of the genetic information to form tubes, branches and entire capillary networks. Specific angiogenic molecules can initiate this process. In view of the physiological importance of angiogenesis, much effort has been devoted to the isolation, characterization and purification of factors that can stimulate angiogenesis. A number of polypeptides which stimulate angiogenesis have been purified and characterized as to their molecular, biochemical and biological properties. For reviews of such angiogenesis regulators, see Klagsbrun et al., "Regulators of Angiogenesis", Ann. Rev. Physiol., 53:217-39 (1991); and Folkman et al., "Angiogenesis," J. Biol. Chem., 267:10931-934 (1992). Recent results have implicated several endothelial receptor tyrosine kinases (RTKs) in the establishment and maintenance of the vascular system.
One such growth factor, which is highly specific as a mitogen for vascular endothelial cells, is termed vascular endothelial growth factor (VEGF). See Ferrara et al., "The Vascular Endothelial Growth Factor Family of Polypeptides," J. Cellular Biochem., 47:211-218 (1991); Connolly, "Vascular Permeability Factor: A Unique Regulator of Blood Vessel Function," J. Cellular Biochem., 47:219-223 (1991). VEGF is a potent vasoactive protein that has been detected in media conditioned by a number of cell lines including bovine pituitary follicular cells. VEGF is a glycosylated cationic 46-48 kD dimer made up of two 24 kD subunits. It is inactivated by sulfhydryl reducing agents, resistant to acidic pH and to heating, and binds to immobilized heparin. VEGF is sometimes referred to as vascular permeability factor (VPF) because it increases fluid leakage from blood vessels following intradermal injection. It also has been called by the name vasculotropin.
Four different molecular species of VEGF have been detected. The 165 amino acid species has a molecular weight of approximately 46 kD and is the predominant molecular form found in normal cells and tissues. A less abundant, shorter form with a deletion of 44 amino acids between positions 116 and 159 (VEGF.sub.121), a longer form with an insertion of 24 highly basic residues in position 116 (VEGF.sub.189), and another longer form with an insertion of 41 amino acids (VEGF.sub.206), which includes the 24 amino acid insertion found in VEGF.sub.189, are also known. VEGF.sub.121 and VEGF.sub.165, are soluble proteins. VEGF.sub.189 and VEGF.sub.206 appear to be mostly cell-associated. All of the isoforms of VEGF are biologically active. For example, each of the species when applied intradermally is able to induce extravasation of Evans blue.
The various species of VEGF are encoded by the same gene and arise from alternative splicing of messenger RNA. This conclusion is supported by Southern blot analysis of human genomic DNA, which shows that the restriction pattern is identical using either a probe for VEGF.sub.165 or one which contains the insertion in VEGF.sub.206. Analysis of genomic clones in the area of putative mRNA splicing also shows an intron/exon structure consistent with alternative splicing.
The different isoforms of VEGF have different chemical properties which may regulate cellular release, compartmentalization, bioavailability and possibly also modulate the signalling properties of the growth factors.
Analysis of the nucleotide sequence of the VEGF gene indicates that VEGF is a member of the platelet-derived growth factor (PDGF) family. VEGF and PlGF are ligands for two endothelial RTKs, flt-1 (VEGF receptor 1, VEGFR1) and flk-1/KDR (VEGF receptor 2, VEGFR2). The amino acid sequence of VEGF exhibits approximately 20% homology to the sequences of the A and B chains of PDGF, as well as complete conservation of the eight cysteine residues found in both mature PDGF chains. VEGF.sub.165, VEGF.sub.189 and VEGF.sub.206 also contain eight additional cysteine residues within the carboxy-terminal region. The amino-terminal sequence of VEGF is preceded by 26 amino acids corresponding to a typical signal sequence. The mature protein is generated directly following signal sequence cleavage without any intervening prosequence. The existence of a potential glycosylation site at Asn.sup.74 is consistent with other evidence that VEGF is glycoprotein, but the polypeptide has been reported to exist in both glycosylated and deglycosylated species.
Like other cytokines, VEGF can have diverse effects that depend on the specific biological context in which it is found. VEGF and its high affinity receptors flt-1 and KDR/flk-1 are required for the formation and maintenance of the vascular system as well as for both physiological and pathological angiogenesis. VEGF is a potent endothelial cell mitogen and directly contributes to induction of angiogenesis in vivo by promoting endothelial cell growth during normal embryonic development, wound healing, and tissue regeneration and reorganization. VEGF is also involved in pathological processes such as growth and metastasis of solid tumors and ischemia-induced retinal disorders. A most striking property of VEGF is its specificity. It is mitogenic in vitro at 1 ng/ml for capillary and human umbilical vein endothelial cells, but not for adrenal cortex cells, corneal or lens epithelial cells, vascular smooth muscle cells, corneal endothelial cells, granulosa cells, keratinocytes, BHK-21 fibroblasts, 3T3 cells, rat embryo fibroblasts, human placental fibroblasts and human sarcoma cells. The target cell specificity of VEGF is thus restricted to vascular endothelial cells. VEGF can trigger the entire sequence of events leading to angiogenesis and stimulates angiogenesis in vivo in the cornea and in a healing bone graft model. It is able to stimulate the proliferation of endothelial cells isolated from both small and large vessels. Expression of VEGF mRNA is temporally and spatially related to the physiological proliferation of capillary blood vessels in the ovarian corpus luteum or in the developing brain. VEGF expression is triggered by hypoxia so that endothelial cell proliferation and angiogenesis appear to be especially stimulated in ischemic areas. VEGF is also a potent chemoattractant for monocytes. In addition, VEGF induces plasminogen activator and plasminogen activator inhibitor in endothelial cells.
Tumor cells release angiogenic molecules such as VEGF, and monoclonal antibodies to VEGF have been shown to inhibit the growth of certain types of tumor such as rhabdomyosarcoma. See Kim et al., "Inhibition of Vascular Endothelial Growth Factor-Induced Angiogenesis Suppresses Tumor Growth in vivo," Nature, 362:841-844 (1993). This suggests that blocking VEGF action is of potential therapeutic significance in treating tumors in general, and highly-vascularized, aggressive tumors in particular.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a new growth factor having the property of promoting proliferation of endothelial cells.
Another object of the invention is to provide isolated DNA sequences which encode a new growth factor which promotes proliferation of endothelial cells.
It is also an object of the invention to provide new products which may be useful in diagnostic and/or therapeutic applications.
These and other objects are achieved in accordance with the present invention by providing an isolated DNA which codes for a protein exhibiting the following characteristic amino acid sequence (SEQ ID NO:16):
Pro-Xaa-Cys-Val-Xaa-Xaa-Xaa-Arg-Cys-Xaa-Gly-Cys-Cys
and having the property of promoting proliferation of endothelial cells or mesodermal cells, the DNA being selected from the group consisting of the DNA of FIGS. 1 and 2 (SEQ ID NO:1), the DNA of FIG. 3 (SEQ ID NO:4), the DNA of FIG. 5 (SEQ ID NO:6); the DNA of FIG. 7 (SEQ ID NO:8), the DNA of FIG. 10 (SEQ ID NO:10), the DNA of FIG. 12 (SEQ ID NO:12), the DNA of FIG. 14 (SEQ ID NO:14), and DNA's which hybridize under stringent conditions with at least one of the foregoing DNA sequences.
In accordance with further aspects of the invention, the objects are also achieved by providing a protein exhibiting the following characteristic amino acid sequence
Pro-Xaa-Cys-Val-Xaa-Xaa-Xaa-Arg-Cys-Xaa-Gly-Cys-Cys
(SEQ ID NO:16) and having the property of promoting proliferation of endothelial cells or mesodermal cells, which protein comprises a sequence of amino acids substantially corresponding to an amino acid sequence selected from the group consisting of the amino acid sequence of FIG. 1 (SEQ ID NO:2), the amino acid sequence of FIG. 2 (SEQ ID NO:3), the amino acid sequence of FIG. 4 (SEQ ID NO:5), the amino acid sequence of FIG. 6 (SEQ ID NO:7), the amino acid sequence of FIG. 8 (SEQ ID NO:9), the amino acid sequence of FIG. 11 (SEQ ID NO:11), the amino acid sequence of FIG. 13 (SEQ ID NO:13), and the amino acid sequence of FIG. 15 (SEQ ID NO:15).
In further aspects of the invention, the objects are achieved by providing pharmaceutical preparations which comprise such proteins; and by providing antibodies which react with or recognize such proteins.
The novel growth factor of the present invention, referred to hereinafter as vascular endothelial growth factor B or VEGF-B, has close structural similarities to VEGF and to placenta growth factor (PlGF). All of the VEGF-B forms contain the characteristic amino acid sequence
Pro-Xaa-Cys-Val-Xaa-Xaa-Xaa-Arg-Cys-Xaa-Gly-Cys-Cys
(SEQ ID NO:16) (wherein Xaa represents a variable residue), which is an earmark of the PDGF/VEGF family of growth factors. This characteristic amino acid sequence can be found at amino acids 70 to 82 in FIGS. 4, 6, 8, 11, 13 and 15.
Clinical applications of the invention include diagnostic applications, acceleration of angiogenesis in wound healing, and inhibition of angiogenesis. Quantitation of VEGF-B in cancer biopsy specimens may be useful as an indicator of future metastatic risk. Topical application of VEGF-B preparations to chronic wounds may accelerate angiogenesis and wound healing. VEGF-B may be used in a manner analogous to VEGF. |
