Vascular Endothelial Growth Factor

Vascular endothelial growth factor was first reported in 1989, and isolated a

endothelial cell specific mitogen from medium condition by bovine pituitary follicular

cells [27]. VEGF is potent, diffusible, and specific for vascular endothelial cells led to

the hypothesis that this factor might play a role in the regulation of physiological and

pathological growth of blood vessels. The role of VEGF in the regulation of

angiogenesis has been investigating in two decades ago [21]. According to published

literature, it has been indicates that new vessel growth are highly complex and

coordinated processes, requiring a serial of ligands binding to numerous receptors,

which led to the proliferation, migration, and angiogenesis of endothelial cells [28].

1.2.5.1 Activities of VEGF

A well known in vitro activity of VEGF is a survival factor for endothelial cells

derived from arteries, veins, and lymphatic. VEGF prevents endothelial apoptosis

induced by serum starvation, which subsequently is mediated by the phosphatidy-

linositol 3-kinase (PI3 kinase)/Akt pathway [29]. Moreover, VEGF could induce

expression of the anti-apoptotic proteins Bcl-2 and A1 in endothelial cells [30].

VEGF induces a potent angiogenic response in a variety of in vivo models and

angiogenesis in tridimensional in vitro models, inducing confluent microvascular

endothelial cells to invade collagen gels and form capillary-like structures [31, 32].

Moreover, VEGF elicits a pronounced angiogenesis response in a variety of in vitro

models including the matrigel plug in mice [33].

1.2.5.2 Tumor associated VEGF

In contrast to normal vessels, vessels in solid tumors are often abnormally enlarged,

and blood flow in tumor vessels is often chaotic, slow and not efficient in meeting

metabolic demands [34]. Furthermore, tumor cells usually represent the main source

of VEGF, though several studies have shown that tumor-associated stroma is also a

site of VEGF production [35, 36]. Many tumor cell lines particularly secrete VEGF in

vitro, suggesting the possibility that this diffusible molecule may be a mediator of

tumor angiogenesis [37]. In situ hybridization studies have demonstrated that VEGF

mRNA is expressed in many tumors, including lung, breast, gastrointestinal tract,

renal, and ovarian carcinomas [32].

1.2.5.3 VEGF gene and isoforms

The VEGF-related gene family of angiogenic and lymphangiogenic growth factors

comprises five secreted glycol- proteins referred to as VEGF-A, VEGF-B, VEGF-C,

VEGF-D, and placenta growth factors (PlGF) [38]. VEGF-A is the beststudied, has

been most strongly associated with angiogenesis,and is the target of most current

anti-VEGF treatments. VEGF-A signals through two receptor tyrosine kinases,

VEGFR1and VEGFR2, and is the only member of the VEGF gene family foundto be

induced by hypoxia [39]. VEGF-B selectively binds to VEGFR1and has a role in the

regulation of extracellular matrix degradation,cell adhesion and migration [40]. Both

VEGF-C and VEGF-D bind to VEGFR-2 and VEGFR-3 and regulate lymph-

angiogenesis, and VEGF-C may alsobe involved in wound healing [41, 42]. PlGF

selectively binds to VEGFR-1 and is the most abundantly expressed VEGF family

member in endothelial cells. PlGF may potentiate VEGF-A-induced endothelialcell

proliferation, but on its own PlGF exerts only weak mitogenicity [39].

VEGF-A is a major angiogenic factor of the VEGF family, and is an important

survival factor for endothelial cells, both in vitro and in vivo. VEGF-A was purified to

be sequenced and cloned by Ferrara and collaborates. Alternative exon splicing of

human VEGFs gene shows that it is comprised eight exons , denoted as: VEGFA121,

VEGFA145, VEGFA165, VEGF165b, VEGFA189 and VEGFA206, gives rise to

isoforms with different length of amino acids, and biological activities [43]. The

properties of native VEGF closely correspond to those of VEGF165. VEGF121 is an

acidic polypeptide that does not bind to heparin. VEGF189 and VEGF206 are highly

basic and bind to heparin with high affinity. Whereas VEGF121 is a freely diffusible

protein, VEGF189 and VEGF206 are almost completely sequestered in the

extracellular matrix (ECM) [38].

VEGFA165 is a heparin-binding homodimeric glycoprotein of 46 kDa.

Structurally, VEGFAs with intra-domain and inter-domain disulfide bonds between

eight cysteine residues conserved positions. Anti-parrallel homodimer covalently

linked by two disulfide bridges between Cys-51 and Cys-60 [44]. VEGFA165 is

secreted, but significant fraction remains bound to the cell surface and extracellular

matrix (ECM), by virtue of its heparin-binding properties. In the present studies,

VEGFA and its receptors is the best characterized signaling pathway in developmental

angiogenesis. Furthermore, much research has also established the VEGFA in tumor

angiogenesis, and VEGFA action constitutes a rate-limiting step in normal and

pathological blood vessel growth [38].

1.2.5.4 Regulation of VEGF gene expression

VEGF gene expression is up-regulated by hypoxia. Hypoxia allows the stabilization

of hypoxia-inducible factors 1 (HIF-1) that binds to specific promoter elements that

are present in the promoter region of VEGFA. This region is a 28-base sequence in the

5’- promoter of human VEGF gene, which mediates hypoxia-induced transcription

[45]. Importantly, another study has implicated the PI3 kinase/Akt pathway in the

regulation of HIF-mediated responses in a hypoxia-independent manner. Mutations in

Akt also results in increased activation of HIF-1 and increased VEGF transcription

[46]. Specific transforming events also result in induction of VEGF gene expression.

Oncogenic mutations or amplification of Ras leads to VEGF up-regulation, which

indicates that mutant Ras-dependent VEGF expression is necessary for progressive

tumor growth in vivo [47]. several major growth factors, including epidermal growth

factor (EGF), TGF-α, TGF-β, FGF and PDGF, also up-regulate VEGF mRNA

expression [48], suggesting that paracrine or autocrine release of such factors

cooperates with local hypoxia in regulating VEGF release in the microenvironment.

Furthermore, inflammatory cytokines such as IL-8 also induce expression of VEGF in

several cell types in agreement with the hypothesis that VEGF may be a mediator of

angiogenesis in inflammatory disorders[49].