Vascular Endothelial Growth Factor Family

VEGF-VEGF R2 Signaling Pathways

VEGF Family Ligands

The Vascular Endothelial Growth Factor (VEGF) family consists of five mammalian proteins, VEGF-A (VEGF), VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF), along with virally encoded VEGF-E, and the snake venom VEGF-F proteins. Members of the VEGF family belong to the VEGF/PDGF cystine knot superfamily and have eight highly conserved cysteine residues at fixed positions. Six of these cysteines form intramolecular disulfide bonds and the remaining two form intermolecular disulfide bonds that stabilize the dimeric structures of the proteins. VEGF family proteins function as potent mitogens for endothelial cells. They are not only required for vasculogenesis and angiogenesis during embryonic development but they are also required for blood vessel growth during pregnancy and wound healing, and for lymphangiogenesis. Additionally, VEGF proteins play a role in the progression of diseases associated with abnormal neovascularization such as proliferative diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, atherosclerosis, and cancer.

VEGF-A  

The VEGF-A gene contains eight exons which are differentially spliced to form at least ten isoforms that are named based on the number of amino acids that they contain. These include VEGF₁₁₀, VEGF₁₁₁, VEGF₁₂₁, VEGF₁₄₅, VEGF₁₄₈, VEGF₁₆₂, VEGF₁₆₅, VEGF_165b, VEGF₁₈₃, VEGF₁₈₉, and VEGF₂₀₆. Of these, VEGF₁₆₅ is the most abundant isoform. Exons 1-5 of VEGF are conserved in all isoforms of the protein. Exon 1 and four amino acids of exon 2 contain the signal peptide, which is cleaved during secretion, while exons 2-5 of VEGF contain the VEGF R1- and VEGF R2-binding domains. Exons 6, 7, and 8a of VEGF are variable in different VEGF isoforms and regulate binding to the VEGF co-receptors, Neuropilin-1, Neuropilin-2, and heparan sulfate. Exons 6 and 7 encode two heparin binding domains, which mediate VEGF binding to heparan sulfate proteoglycans (HSPGs) on the cell surface or in the extracellular matrix, while exon 8a encodes the short, C-terminal Neuropilin-binding domain. VEGF isoforms that contain exon 8b rather than exon 8a are unable to interact with the Neuropilin receptors. All isoforms of VEGF are secreted, but the larger isoforms that contain exons 6 and 7, such as VEGF₁₈₃, VEGF₁₈₉, and VEGF₂₀₆, may be sequestered in the extracellular matrix and require plasmin-mediated proteolysis to become diffusible. VEGF functions as a pro-angiogenic factor by promoting endothelial cell proliferation, survival, migration and tube formation, and increasing vascular permeability. It is highly expressed during development, and mice lacking even one copy of the VEGF-A gene die during early embryogenesis. VEGF expression declines after birth and is relatively low in most adult tissues, but it is up-regulated during pregnancy, wound healing, and under conditions associated with hypoxia, hypoglycemia, mechanical stress, and inflammation.

VEGF-B

In addition to the different isoforms of VEGF, two alternatively spliced isoforms of VEGF-B, VEGF-B₁₆₇ and VEGF-B₁₈₆ have also been reported. The VEGF-B gene contains seven exons. The first five exons are conserved in both VEGF-B₁₆₇ and VEGF-B₁₈₆, but alternative splicing in exon 6 generates two proteins with different carboxy-terminal ends. While VEGF-B₁₆₇ contains a highly basic cysteine-rich heparin-binding domain in its C-terminus that binds the protein to the cell surface or sequesters it in the extracellular matrix after it is secreted, VEGF-B₁₈₆ lacks this domain and is diffusible. Unlike VEGF, which promotes angiogenesis by stimulating endothelial cell proliferation and vascular permeability, the function of VEGF-B in angiogenesis is not currently well understood. While VEGF-B does not act as a potent mitogen for endothelial cells and is not required for embryonic angiogenesis, it has been found to be expressed in most tumors and tumor-derived cell lines. Additionally, it has been shown to inhibit apoptosis of a broad range of cells, including vascular cells, cardiac myocytes, and neurons.

VEGF-C and VEGF-D

VEGF-C and VEGF-D are structurally and functionally related proteins. They are both expressed as inactive propeptides that contain long N- and C-terminal extensions flanking the VEGF homology domain. Two sequential proteolytic processing events are necessary to generate the mature, biologically active VEGF-C and VEGF-D proteins. Both VEGF-C and VEGF-D promote lymphangiogenesis by binding with high affinity to VEGF R3, which is primarily expressed on lymphatic endothelial cells. Additionally, both VEGF-C and VEGF-D can bind with weak affinity to VEGF R2 and promote a low degree of angiogenesis. VEGF-C is expressed during embryogenesis and is essential for the development of the lymphatic system in mice. In contrast, VEGF-D is expressed in adult tissues including the heart, lung, and small intestine, and is not required for embryonic development.

PlGF

Placental growth factor (PlGF) is another member of the VEGF family. Similar to VEGF and VEGF-B, four isoforms of human PlGF can be generated by alternative splicing. These isoforms are known as PlGF-1, PlGF-2, PlGF-3, and PlGF-4. In humans, PlGF-1 and PlGF-2 are thought to be the major isoforms of PlGF, while only PlGF-2 has been identified in mice. Human PlGF-1 and PlGF-2 consist of 131 and 152 amino acids, respectively. In contrast to PlGF-1, PlGF-2 contains a highly basic heparin-binding insert at its C-terminus and is able to bind to both heparin and Neuropilin-1 through this domain. PlGF-3 is similar to PlGF-1 in that it lacks the C-terminal heparin-binding domain, but in addition, it contains a 216-nucleotide insertion between exons 4 and 5. PlGF-4 has this 216-nucleotide insertion as well, but like PlGF-2, it also contains the heparin-binding insert at its C-terminus.

PlGF signals through VEGF R1/Flt-1, but not VEGF R2/KDR/Flk-1 and it can affect angiogenesis both directly and indirectly. PlGF competes with VEGF for binding to VEGF R1/Flt-1, which reduces VEGF-VEGF R1 binding and thereby promotes VEGF-VEGF R2 signaling and angiogenesis. In contrast, PlGF can also heterodimerize with some forms of VEGF, which decreases the angiogenic effects of VEGF-VEGF R2 signaling. PlGF can also affect angiogenesis indirectly by promoting the activation and/or recruitment of pro-angiogenic cell types, including myeloid progenitors and macrophages, which produce inflammatory cytokines and angiogenic molecules. In mice, PlGF is not required for embryonic development or reproduction, but postnatally, these mice display defects in angiogenesis in response to ischemia.

VEGF Receptors

The biological effects of different VEGF family ligands are mediated by binding to one of three receptors belonging to the class III subfamily of receptor tyrosine kinases: VEGF R1/Flt-1, VEGF R2/KDR/Flk-1, and VEGF R3/Flt-4. These receptors are all single-pass transmembrane proteins with seven extracellular Ig-like domains and a split intracellular tyrosine kinase domain. VEGF receptors are primarily expressed on endothelial cells, but they each mediate distinct biological effects. VEGF binding to VEGF R2 is primarily responsible for stimulating endothelial cell proliferation, migration, and tube formation associated with angiogenesis, while VEGF-C and VEGF-D promote lymphangiogenesis by binding to VEGF R3. In contrast to VEGF R2, VEGF R1 has been characterized as a decoy receptor that negatively regulates VEGF signaling by preventing VEGF from binding to VEGF R2. Although VEGF binds to VEGF R1 with higher affinity than it binds to VEGF R2, the tyrosine kinase activity of VEGF R1 is about ten-fold weaker than that of VEGF R2 and multiple studies have shown that the pro-angiogenic effects of VEGF are primarily attributed to its interaction with VEGF R2. As previously described, some forms of VEGF can also bind to heparan sulfate proteoglycans and to Neuropilin-1 or Neuropilin-2. The Neuropilins and HSPG function as co-receptors to regulate VEGF ligand/receptor interactions and can enhance or modulate downstream signaling.

VEGF Signaling

Binding of VEGF family ligands to the VEGF receptors triggers receptor dimerization and autophosphorylation of its intracellular tyrosine kinase domain. For VEGF R2, this leads to the recruitment of numerous intracellular signaling molecules, such as GRB2, Shb, Src, PLC-gamma, PI 3-kinase, NCK/Fyn, CDC42, and SH2D2A, which results in activation of multiple downstream signaling pathways. These include the PI 3-kinase/Akt pathway, the Ras-MAPK pathway, the Shb/FAK/Paxillin pathway, the PLC-gamma/PKC pathway, and the PAK2/CDC42/p38 MAPK pathway. Together, these pathways promote the cellular effects of VEGF including endothelial cell migration, survival, proliferation, and vascular permeability to drive angiogenesis.