**4. Vascular endothelial growth factor (VEGF)**

Vascular endothelial growth factor (VEGF, now referred to as VEGF-A) is a member of a family of proteins including VEGF-B, VEGF-C, VEGF-D, VEGF-E (virally encoded), and PlGF. VEGF-C and VEGF-D are primarily implicated in regulation of lymph angiogenesis. Given the dominant role that VEGF-A plays in regulating angiogenesis and disease, it will be referred to as VEGF. VEGF undergoes multiple splicing alternative creating several exon leading to multiple isoforms. Common isoforms include VEGF 165, VEGF 206, VEGF121, and VEGF189. VEGF165 (VEGF164 in mice) is the most frequently expressed isoform in majority of tissues. The VEGF165 is also the most physiologic isoform, with characteristics connected to the highly diffusible VEGF121 and the extracellular matrix (ECM)-bound VEGF189 [32, 33].

Less other isoforms of VEGF, such as VEGF145 and VEGF183 currently been described in several studies. Main features differentiates one isoforms than another were differential ability to bind heparin. The lowest affinity to heparin belongs to VEGF121, while strong affinity known for VEGF189 and VEGF206 which consist of two heparin-binding domains (encoded by exons 6 and 7), that may also bind to protein in the cell surfaces or the ECM. The most common VEGF165 has an intermediate binding ability with a single heparin-binding domain, encoded by exon 7, and has ability for ECM bound. In inflammatory process such as appendicitis, several proinflammatory molecules with protease ability such as the MMP3 and plasmin may alter the binding site of VEGF primarily at the COOH terminus and turns VEGF from ECM-bound peptides into non-heparin-binding, diffusible, molecular species which leads to less ability inducing angiogenesis [32].

Several inhibitory isoforms of VEGF have also been recently described, including VEGF165b and VEGFAx, but there is some controversy regarding the mechanisms of inhibition, and VEGF-Ax has now been shown to actually have pro-angiogenic and pro-permeability features. VEGF expression is majorly regulated by the hypoxia state by a transcription factor named hypoxia-inducible factor (HIF). The HIF and other genes related and activated by hypoxia plays role in diverse contexts activating several transcription of other growth factors including platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and some oncogenic gene mutations (RAS, VHL, WNT-KRAS signaling pathway genes) which may control the VEGF expression in other side alters the VEGF-driven signaling [31].

The most understood VEGF signaling now is through VEGFR1/R2 regulation which controls the activities of several kinases and activation of its cascades to promote cell proliferation, survival, migration, and even influencing vascular permeability on angiogenesis. The endothelial cell, which consist of both tip and stalk cells are at the main site of vascular proliferation. VEGF gradients induce tip cells and promote the formation of filopodia. The molecular regulation of these events is via activation of notch signaling and by increased expression of notch ligands on endothelial cells, including but not limited to delta-like 4 (DLL4). The increased signaling of the notch in neighboring cells will reduces the expression of VEGFR2, which is causing a negative feedback loop to the signaling process. This main signaling pathway of the VEGF plays a critical role to maintain homeostasis, but as alteration of the pathway lead to hyperactivation by pathologic process leading to pathologic angiogenesis. Another pathway were described in 2014, named as a non-canonical pathway of VEGFR2 that was characterized in neurons. It is known to be expressed more in retinal neurons but are lacking in endothelial cells. Study reveals that a deletion gene responsible in

#### **Figure 4.**

*VEGF activation and signaling pathways [33].*

VEGFR2 pathway in neurons causes abnormal angiogenesis process by high VEGF expressions around the neuron tissue in response to deficiency of the VEGFR2. In other hand, the abnormal angiogenesis at the juxta-neural cells were common in response to maintain homeostasis in cases of ischemic retinopathy to ensure regenerative phase. This similar mechanism were a point of interest as number of VEGF expressed would be a critical factor to maintain tissue vascularization in several pathogenesis of tissue damage (**Figure 4**) [31, 33].

The findings that anti-VEGF antibodies decreased the growth of tumor cells implanted in immune-deficient mice opened up translational possibilities for targeting VEGF-VEGFR signaling. In addition, it was also demonstrated that inactivation of a single allele of the VEGF-A gene in mice resulted in defective vascular development and early embryonic lethality, highlighting the importance of VEGF during embryonic development. Inactivation of both copies of vegfr2 largely pheno-copied vegfa single-allele deletion. The ability to delete VEGF in target tissues with the advent of cre-lox systems created the possibility of assessing the role of VEGF in individual

tissues/cells. Numerous studies employing this approach have documented the important role of VEGF in angiogenesis and homeostasis in a variety of pathophysiological circumstances [34].
