**3. The VEGF gene and isoforms**

The VEGFs term identifies a large and heterogeneous family of secreted polypeptides, named VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placenta growth factor (PGF), characterized by a highly conserved receptor-binding cystine-knot structure similar to that of the platelet-derived growth factors [13, 14]. Of those six members, VEGF-A plays a key role in vasculogenesis and angiogenesis [15]. PGF is mainly expressed in placenta, heart and lungs; it is a ligand for VEGFR-2 and it is involved in angiogenesis regulation. VEGF-B binds VEGFR-1 and neuropilin-1, mostly expressed in the extracellular matrix and abundantly expressed in brown fat, in the myocardium and skeletal muscle; it is implicated in high cellular energy metabolism. VEGF-C is produced as a precursor protein [16, 17]. VEGF-C promotes mitogenesis, migration and survival of ECs [18], and regulates the lymphatic vessel growth by binding to VEGF-receptor-3 (VEGFR-3, Flt-4) [19, 20]. VEGF-D, binds and activates VEGFR-2 and VEGFR-3, because is mitogenic for EC, angiogenic, and lymphangiogenic. Its expression was demonstrated mainly in the lung and skin during embryogenesis. VEGF-E expression, binding the VEGFR-2, promotes the release of tissue factors, proliferation, chemotaxis and

**123**

*Vascular Endothelial Growth Factor Expression in the Pathological Angiogenesis in Oral…*

sprouting of cultured vascular ECs in vitro and angiogenesis in vivo [6, 21]. The major function of VEGF-C consist in regulation VEGF exerts its effect on tissues at several levels; mechanisms range from a plethora of physiological processes which regulate blood vessel growth, such as during pregnancy and in tissue repair to pathological conditions, including chronic inflammation, would healing process, and cancer [6, 22]. It has been proven that VEGF causes a pronounced angiogenic response in a variety of in vivo models, including the chick chorioallantoic membrane [23, 24]. Native VEGF is a basic, heparin-binding, homodimeric glycoprotein of 45,000 daltons. Interestingly, the VEGF gene has been mapped to chromosome 6 at position p21.3 [25] and consists of eight exons and seven introns in the coding sequence, which covers a region of 14 kilobases. Among these, there is evidence that the −634G/C, −1154G/A, and −2578C/A *VEGF* polymorphisms have been shown to be associated with increased VEGF production. Alternative splicing of the mRNA from the gene of VEGF, VEGF-B, and PIGF results in the expression of five known human isoforms with differential diffusibility and heparin-binding properties containing 121, 145, 165, 183, 189 and 206 amino acids with different biological properties: VEGF121, VEGF165, VEGF189, and VEGF206 [26]. The VEGF121 is a weakly acidic polypeptide with a 44 *amino-acid insertion* encoded by exon 7a*,* that fails to bind to heparin [27]; *VEGF189 has* a further *insertion* of 24 *amino acids, highly enriched in basic residues* encoded by exon 6a. VEGF121 and VEGF165 have been detected predominantly in normal tissue, but VEGF121 isoform is both more angiogenic and tumorigenic than being the 165 and 189 isoforms [27, 28]. In particular, the isoform 121 has been shown to predominate in primary human breast carcinomas (Relf M) VEGF189 expression has been shown to be dominant in normal lung [29] and the 183 isoform predominates in heart [30, 31]. Several studies have reported that the

The VEGF and the "fibroblastic growth factor" (bFGF) demonstrate a powerful synergism in the promotion of angiogenesis in vitro, as shown by models using microvascular endothelial cells invading the *three*-*dimensional collagen* gel system [32, 33]. Also, VEGF exerts its effect by coordinating of angiopoietins, another class of angiogenic factors. Specifically, VEGF is involved in the early sequences of events leading to the vessel development, whereas angiopoietin 1 (Ang1), an agonist ligand for the endothelial-specific Tie2 receptor, binds and activates Tie2 to promote vessel maturation, vascular stability and leakiness [34]; Ang2 acts as a Tie2 agonist in lymphatic endothelial cells generating an important vascular signaling pathway involved in angiogenesis, vascular stability and quiescence. VEGF achieves its functions of endothelial cell differentiation and proliferation by binding a family of tyrosine kinases receptors (VEGFRs), known as Flt-1 receptor (VEGF receptor-1) and VEGF receptor-2 (VEGF-2 or KDR/Flk-1). VEGFR-1 binds VEGF, VEGF-B and PlGF with high affinity and induces weak mitogenic signals in ECs. [23, 35] VEGFR-1 expression is up-regulated by hypoxia via (transcription hypoxia inducible factor) HIF-dependent mechanism [36, 37]. In the lung, VEGFR-1 provokes secretion of Matrix Metalloproteinase 9 (MMP9) at the vascular bed, thus empowering metastasis. Also, it has been discovered the role of VEGFR-1 in releasing tissue specific factors in a perivascular specific pattern at the level of vascular endothelium [38]. VEGFR-2, the major mediator of endothelial cell mitogenesis, proliferation and survival [39, 40] binds VEGF, VEGF-C, VEGF-D, VEGF-E and PlGF [22, 39]. VEGFR-2 expression is down-regulated in the adult blood vascular ECs, and is again up-regulated in the endothelium of angiogenic process. VEGF, VEGF-C and VEGF-D are bound VEGFR-3 and are involved in regulation of lymphangiogenesis,

*DOI: http://dx.doi.org/10.5772/intechopen.90924*

expression of VEGF206 occurs mainly in fetal liver.

**3.1 Biological effects of VEGF expression**

*Vascular Endothelial Growth Factor Expression in the Pathological Angiogenesis in Oral… DOI: http://dx.doi.org/10.5772/intechopen.90924*

sprouting of cultured vascular ECs in vitro and angiogenesis in vivo [6, 21]. The major function of VEGF-C consist in regulation VEGF exerts its effect on tissues at several levels; mechanisms range from a plethora of physiological processes which regulate blood vessel growth, such as during pregnancy and in tissue repair to pathological conditions, including chronic inflammation, would healing process, and cancer [6, 22]. It has been proven that VEGF causes a pronounced angiogenic response in a variety of in vivo models, including the chick chorioallantoic membrane [23, 24]. Native VEGF is a basic, heparin-binding, homodimeric glycoprotein of 45,000 daltons. Interestingly, the VEGF gene has been mapped to chromosome 6 at position p21.3 [25] and consists of eight exons and seven introns in the coding sequence, which covers a region of 14 kilobases. Among these, there is evidence that the −634G/C, −1154G/A, and −2578C/A *VEGF* polymorphisms have been shown to be associated with increased VEGF production. Alternative splicing of the mRNA from the gene of VEGF, VEGF-B, and PIGF results in the expression of five known human isoforms with differential diffusibility and heparin-binding properties containing 121, 145, 165, 183, 189 and 206 amino acids with different biological properties: VEGF121, VEGF165, VEGF189, and VEGF206 [26]. The VEGF121 is a weakly acidic polypeptide with a 44 *amino-acid insertion* encoded by exon 7a*,* that fails to bind to heparin [27]; *VEGF189 has* a further *insertion* of 24 *amino acids, highly enriched in basic residues* encoded by exon 6a. VEGF121 and VEGF165 have been detected predominantly in normal tissue, but VEGF121 isoform is both more angiogenic and tumorigenic than being the 165 and 189 isoforms [27, 28]. In particular, the isoform 121 has been shown to predominate in primary human breast carcinomas (Relf M) VEGF189 expression has been shown to be dominant in normal lung [29] and the 183 isoform predominates in heart [30, 31]. Several studies have reported that the expression of VEGF206 occurs mainly in fetal liver.

#### **3.1 Biological effects of VEGF expression**

The VEGF and the "fibroblastic growth factor" (bFGF) demonstrate a powerful synergism in the promotion of angiogenesis in vitro, as shown by models using microvascular endothelial cells invading the *three*-*dimensional collagen* gel system [32, 33]. Also, VEGF exerts its effect by coordinating of angiopoietins, another class of angiogenic factors. Specifically, VEGF is involved in the early sequences of events leading to the vessel development, whereas angiopoietin 1 (Ang1), an agonist ligand for the endothelial-specific Tie2 receptor, binds and activates Tie2 to promote vessel maturation, vascular stability and leakiness [34]; Ang2 acts as a Tie2 agonist in lymphatic endothelial cells generating an important vascular signaling pathway involved in angiogenesis, vascular stability and quiescence. VEGF achieves its functions of endothelial cell differentiation and proliferation by binding a family of tyrosine kinases receptors (VEGFRs), known as Flt-1 receptor (VEGF receptor-1) and VEGF receptor-2 (VEGF-2 or KDR/Flk-1). VEGFR-1 binds VEGF, VEGF-B and PlGF with high affinity and induces weak mitogenic signals in ECs. [23, 35] VEGFR-1 expression is up-regulated by hypoxia via (transcription hypoxia inducible factor) HIF-dependent mechanism [36, 37]. In the lung, VEGFR-1 provokes secretion of Matrix Metalloproteinase 9 (MMP9) at the vascular bed, thus empowering metastasis. Also, it has been discovered the role of VEGFR-1 in releasing tissue specific factors in a perivascular specific pattern at the level of vascular endothelium [38]. VEGFR-2, the major mediator of endothelial cell mitogenesis, proliferation and survival [39, 40] binds VEGF, VEGF-C, VEGF-D, VEGF-E and PlGF [22, 39]. VEGFR-2 expression is down-regulated in the adult blood vascular ECs, and is again up-regulated in the endothelium of angiogenic process. VEGF, VEGF-C and VEGF-D are bound VEGFR-3 and are involved in regulation of lymphangiogenesis,

*Oral Diseases*

of oral cancer development.

**2. Discovery of VEGF**

domain [11, 12].

**3. The VEGF gene and isoforms**

of the vascular development of tumor xenografts in mice. These results were confirmed through in situ hybridization studies, showing a correlation between the degree of defective angiogenesis and VEGF mRNA upregulation. These studies uniformly concur that VEGF expression enhances tumor growth. In this chapter, we provide a brief historical overview of the discovery of VEGF, structural characterization of the other members of VEGF family and their receptor, and to summarize the main features of the role of vascular endothelial growth factor in angiogenesis

VEGF was first described in 1983 as a factor secreted by hepatocarcinoma cell lines [1] that increased microvascular permeability to plasma proteins in the skin of guinea-pigs. It was highly purified to homogeneity from pituitary folliculostellate cells and characterized in 1989 [2]. Other authors supported evidence that this protein potently stimulated endothelial cell migration [3–5]. It was named "vascular permeability factor" or VPF, potent inducers of vascular hyperpermeability (especially venular endothelium) to fibrinogen and other plasma proteins [6], which upon secretion by tumor cells, promotes vascular leakage [7]. More years later, it was demonstrated that VPF has potent mitogenic activity in a diversity of cell types, and also versus endothelial cells [8]. Vascular endothelial growth factors (VEGFs) are predominantly produced by endothelial, hematopoietic, and stromal cells in response to hypoxia and upon stimulation by growth factors such as transforming growth factor β (TGFβ), interleukins, or platelet-derived growth factors (PDGFs). VEGFs specifically interact with one or several receptor tyrosine kinases (RTKs), VEGF receptor −1, −2, and −3 (VEGFR-1, −2, −3), and with distinct coreceptors such as neuropilins or heparan sulfate glycosaminoglycans. VEGF receptors are classified as type V RTKs whose extracellular domains consist of seven immunoglobulin-like (Ig-like) domains [9, 10]. The intracellular domain consists of seven immunoglobulin-like domains (I–VII), a single transmembrane (TM) region, and a tyrosine kinase consensus sequence (TK) interrupted by a cytoplasmatic kinase

The VEGFs term identifies a large and heterogeneous family of secreted polypeptides, named VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placenta growth factor (PGF), characterized by a highly conserved receptor-binding

cystine-knot structure similar to that of the platelet-derived growth factors [13, 14]. Of those six members, VEGF-A plays a key role in vasculogenesis and angiogenesis [15]. PGF is mainly expressed in placenta, heart and lungs; it is a ligand for VEGFR-2 and it is involved in angiogenesis regulation. VEGF-B binds VEGFR-1 and neuropilin-1, mostly expressed in the extracellular matrix and abundantly expressed in brown fat, in the myocardium and skeletal muscle; it is implicated in high cellular energy metabolism. VEGF-C is produced as a precursor protein [16, 17]. VEGF-C promotes mitogenesis, migration and survival of ECs [18], and regulates the lymphatic vessel growth by binding to VEGF-receptor-3 (VEGFR-3, Flt-4) [19, 20]. VEGF-D, binds and activates VEGFR-2 and VEGFR-3, because is mitogenic for EC, angiogenic, and lymphangiogenic. Its expression was demonstrated mainly in the lung and skin during embryogenesis. VEGF-E expression, binding the VEGFR-2, promotes the release of tissue factors, proliferation, chemotaxis and

**122**

the growth of new lymphatic vessels [41–45]. The expression of VEGFR-3 (or Flt-4) is relevant in lymphatic vessels [44] and in hematopoietic cells of monocytic lineage [6, 42], and is also expressed in a subset of capillary endothelia [19]. Studies on animal models showed that VEGF-C/VEGFR-3 axis plays a crucial role in cancer metastasis by inducing lymphangiogenesis [46–48], but further investigations would be necessary. Furthermore, it has been documented the link between mutations in VEGFR-3 with hereditary lymphedema, an autosomal dominant disorder of the lymphatic system that can lead to lymphangiosarcomas [13, 49]. Neuropilins-1 and -2 are more important in immunology and neuronal development, but they are also involved in angiogenesis [19, 44]. Neuropilins, bind especially class 3 semaphorins but the Neuropilin-1 also binds VEGF, VEGF-B and PlGF, while Neuropilin-2 binds VEGF, VEGF-C and PlGF [50]. When is coexpressed in cells together with VEGFR-2, Neuropilin-1 enhances the binding of VEGF165 to VEGFR-2 and augments tumor angiogenesis in vivo [51, 52]. Nrp-2 is expressed also on lymphatic ECs, and mutated Nrp-2 forms induce abnormalities in the formation of small lymphatic vessels and lymphatic capillaries in mice [53]. In addition, some isoforms bind to known as non-tyrosine kinase receptors, known as neuropilins (NRPs) (neuropilin-1 and neuropilin-2 (22–44)). Neuropilins-1 and -2 are also involved in immunology and neuronal development [50, 54]. Neuropilins bind, especially class 3 semaphorins but the Neuropilin-1 also binds VEGF, VEGF-B and PlGF, while Neuropilin-2 binds VEGF, VEGF-C and PlGF [50]. Neuropilin-1 is capable to improve the binding of VEGF165 to VEGFR-2 and increase tumor angiogenesis in vivo [51, 53]. Nrp-2 is expressed also on lymphatic ECs, and it has been shown in vivo that mutated Nrp-2 form alters the formation of small lymphatic vessels and lymphatic capillaries in mice [54]. The binding results in stimulation of cell-signaling pathways that act to increase cell nucleus division, and contributing to angiogenesis through extracellular matrix dissolution, and endothelial cell movement.
