13.1Vascular Endothelial Growth Factor and Its Functions in the Retina

vascularization of the embryo, as even the knockout of one single allele is lethal for the developing organism [1]. Precise regulation of VEGF is imperative for development, as that not only deletion but also modest overexpression results in embryonal lethality [2]. VEGF was identified in 1983 (termed Vascular Permability Factor) [3] and in 1989 [4] and cloned shortly thereafter [5]. VEGF is expressed in different isoforms due to alternative splicing of the VEGF gene. The gene consists of 8 exons and 7 introns, and all isoforms consist of introns 1–5 and 8 while differing in the splicing of exons 6 and 7 [6]. The isoforms are designated by their number of amino acids, consisting of VEGF121, VEGF145 [7], VEGF148 [8], VEGF165, VEGF183 [9], VEGF189, and VEGF206. A proteolysis-resistent isoform that is induced by genotoxic agents has also been described [10]. The isoforms differ in their ability to bind to heparan sulfate proteoglycans (HS), which results in a different solubility (hence availability) of the isoforms, the smallest being the most soluble. The longer isoforms, VEGF189 and VEGF206, are sequestered to the extracellular matrix (ECM) but can be released by plasmin as a bioactive VEGF110 isoform or by matrix metalloprotease (MMP)-3 as VEGF113 [11]. VEGF165, which has intermediate binding properties, can also bind to and be released from the ECM [12, 13]. The importance of the longer isoforms and their activation by plasmin in pathological angiogenesis is not fully understood so far [14]. Also, the isoforms differ in their ability to bind to the neuropilin (NP) coreceptors [15]. The most abundant VEGF isoforms in the retina are VEGF121 and VEGF165 [16]. Additionally, the expression of VEGF189 has been described [17], and VEGF183 is expressed in retinal Müller cells [18]. VEGF145, VEGF148, or VEGF206 have not been described in the retina so far. Additionally, inhibitory, antiangiogenic isoforms are expressed. They differ in their C-terminal

amino acid residues, designated as VEGFxxxb [19, 20] and are able to inhibit experimental choroidal neovascularization (CNV) [21]. They can bind to Vascular

Endothelial Growth Factor Receptor (VEGFR)-2 and inhibit the downstream signaling pathway (discussed in detail below) [20]. VEGFxxxb isoforms are expressed in the retina, in the retinal pigment epithelium (RPE) and choroid, VEGF165b being the

most abundant [21]. In fact, expression of up to 74% of VEGFxxxb isoforms has been claimed for unstimulated RPE cells [22].

The molecular mechanisms controlling alternative splicing of the VEGF isoforms has not been fully elucidated so far. Serine–arginine rich proteins (SR) which are involved in the regulation of the splicing of eukaryotic mRNA, have been discussed to participate in VEGF splicing [23]. Acidosis induces VEGF121 which is accompanied by an increase in several SR proteins (SF2/ASF, Srp20, Srp40) [24]. The expression of antiangiogenic isoforms could be increased by transforming growth factor beta (TGFb)1 via a Mitogen-activated protein kinase (MAPK) p38, CDC-like kinase (CLK1), and SRp55 dependent pathway, while Insulin-like growth factor (IGF)1 and TNFa increased the angiogenic isoforms via ASF/SF2 and SRp40. SRp55 binds to the 3¢ untranslated region (UTR) region and seems to be the determinant factor for the expression of antiangiogenic VEGF [22]. The transcription factor E2F1, which downregulates the activity of the VEGF promoter under hypoxic conditions [25], is also involved in the shift from angiogenic to antiangiogenic VEGF isoforms. It activates the SR protein SC35, which increases the