First printed in R&D Systems' 2000 Catalog.
Angiogenesis is a process of new blood vessel development from pre-existing vasculature. It plays an essential role in embryonic development, normal growth of tissues, wound healing, the female reproductive cycle (i.e., ovulation, menstruation and placental development), as well as a major role in many diseases.1 Particular interest has focused on cancer, since tumors cannot grow beyond a few millimeters in size without developing a new blood supply. Angiogenesis is also necessary for the spread and growth of tumor cell metastases.2,3
One of the most important growth and survival factors for endothelium is vascular endothelial growth factor (VEGF).4 VEGF induces angiogenesis and endothelial cell proliferation and it plays an important role in regulating vasculogenesis. VEGF is a heparin-binding glycoprotein that is secreted as a homodimer of 45 kDa.5-7 Most types of cells, but usually not endothelial cells themselves, secrete VEGF. Since the initially discovered VEGF, VEGF-A, increases vascular permeability, it was known as vascular permeability factor.8 In addition, VEGF causes vasodilatation, partly through stimulation of nitric oxide synthase in endothelial cells.9 VEGF can also stimulate cell migration and inhibit apoptosis.10
There are several splice variants of VEGF-A. The major ones include: 121, 165, 189, and 206 amino acids (aa), each one comprising a specific exon addition. VEGF165 is the most predominant protein, but transcripts of VEGF may be more abundant.11 VEGF is rarely expressed and has been detected only in fetal liver. Recently, other splice variants of 145 and 183 aa have also been described.12,13 The 165, 189, and 206 aa splice variants have heparin-binding domains, which help anchor them in extracellular matrix and are involved in binding to heparin sulfate and presentation to VEGF receptors. This is a key factor for VEGF potency (i.e., the heparin-binding forms are more active). Several other members of the VEGF family have been cloned including VEGF-B, -C, and -D. 14,15 Placenta growth factor (PlGF) is also closely related to VEGF-A. VEGF-A, -B, -C, -D, and PlGF are all distantly related to platelet-derived growth factors-A and -B. Less is known about the function and regulation of VEGF-B, -C, and -D, but they do not seem to be regulated by the major pathways that regulate VEGF-A.
VEGF-A transcription is potentiated in response to hypoxia and by activated oncogenes. The transcription factors, hypoxia inducible factor-1 alpha (HIF-1 alpha, are degraded by proteasomes in normoxia and stabilized in hypoxia.16,17 This pathway is dependent on the Von Hippel-Lindau gene product.18 HIF-1 alpha and HIF-2 alpha heterodimerize with the aryl hydrocarbon nuclear translocator in the nucleus and bind the VEGF promoter/enhancer. This is a key pathway expressed in most types of cells. Hypoxia inducibility, in particular, characterizes VEGF-A vs. other members of the VEGF family and other angiogenic factors. VEGF transcription in normoxia is activated by many oncogenes including H-ras and several transmembrane tyrosine kinases such as the epidermal growth factor receptor and ErbB2. 19-21 These pathways together account for a marked upregulation of VEGF-A in tumors compared to normal tissues and are often of prognostic importance.22
|Figure 1. Receptors for VEGF and related ligands include: VEGF R1 (Flt-1), VEGF R2 (KDR/Flk-1), VEGF R3 (Flt-4), Neuropilin-1, and Neuropilin-2. The interaction of heparin-binding forms of VEGF with heparan sulfate may assist in presentation to VEGF receptors. For ligand-receptor binding specificities, refer to the chart below.
There are three receptors in the VEGF receptor family.23,24 They have the common properties of multiple IgG-like extracellular domains and tyrosine kinase activity. The enzyme domains of VEGF receptor 1 (VEGF R1, also known as Flt-1), VEGF R2 (also known as KDR or Flk-1), and VEGF R3 (also known as Flt-4) are divided by an inserted sequence (see figure 1). Endothelial cells also express additional VEGF receptors, Neuropilin-1 and Neuropilin-2. 25 VEGF-A binds to VEGF R1 and VEGF R2 and to Neuropilin-1 and Neuropilin-2. 25,26 PlGF and VEGF-B bind VEGF R1 and Neuropilin-1.25,27,28 VEGF-C and -D bind VEGF R3 and VEGF R2.
The VEGF-C/VEGF R3 pathway is important for lymphatic proliferation.29 VEGF R3 is specifically expressed on lymphatic endothelium. A soluble form of Flt-1 can be detected in peripheral blood and is a high affinity ligand for VEGF.30 Soluble Flt-1 can be used to antagonize VEGF function. VEGF R1 and VEGF R2 are upregulated on tumor and proliferating endothelium, partly by hypoxia and also in response to VEGF-A itself. VEGF R1 and VEGF R2 can interact with multiple downstream signaling pathways via proteins such as PLC-gamma, Ras, Shc, Nck, PKC and PI3-kinase.31 VEGF R1 is of higher affinity than VEGF R2 and mediates motility and vascular permeability. VEGF R2 is necessary for proliferation.
VEGF can be detected in both plasma and serum samples of patients, with much higher levels in serum.32 Platelets release VEGF upon aggregation and may be a major source of VEGF delivery to tumors.33 Several studies have shown that association of high serum levels of VEGF with poor prognosis in cancer patients may be correlated with an elevated platelet count.34 Many tumors release cytokines that can stimulate the production of megakaryocytes in the marrow and elevate the platelet count. This can result in an indirect increase of VEGF delivery to tumors.35
VEGF is implicated in several other pathological conditions associated with enhanced angiogenesis. For example, VEGF plays a role in both psoriasis and rheumatoid arthritis.36 Diabetic retinopathy is associated with high intraocular levels of VEGF. Inhibition of VEGF function may result in infertility by blockade of corpus luteum function.37 Direct demonstration of the importance of VEGF in tumor growth has been achieved using dominant negative VEGF receptors38 to block in vivo proliferation, as well as blocking antibodies to VEGF39 or to VEGF R2. Interference with VEGF function has therefore become of major interest for drug development to block angiogenesis. Approaches include antagonists of VEGF or its receptors, selective tyrosine kinase inhibitors,40 targeting of drugs and toxins to VEGF receptors,41 and gene therapy regulated by the same hypoxia pathway that controls VEGF production.42 Targeting the VEGF signaling pathway may be of major therapeutic importance for many diseases.
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