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Microcirculation. Author manuscript; available in PMC 2010 Aug 29.
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PMCID: PMC2929464

Molecular Diversity of VEGF-A as a Regulator of Its Biological Activity


The vascular endothelial growth factor (VEGF) family of proteins regulates blood flow, growth, and function in both normal physiology and disease processes. VEGF-A is alternatively spliced to form multiple isoforms, in two subfamilies, that have specific, novel functions. Alternative splicing of exons 5–7 of the VEGF gene generates forms with differing bioavailability and activities, whereas alternative splice-site selection in exon 8 generates proangiogenic, termed VEGFxxx, or antiangiogenic proteins, termed VEGFxxxb. Despite its name, emerging roles for VEGF isoforms on cell types other than endothelium have now been identified. Although VEGF-A has conventionally been considered to be a family of proangiogenic, propermeability vasodilators, the identification of effects on nonendothelial cells, and the discovery of the antiangiogenic subfamily of splice isoforms, has added further complexity to their regulation of microvascular function. The distally spliced antiangiogenic isoforms are expressed in normal human tissue, but downregulated in angiogenic diseases, such as cancer and proliferative retinopathy, and in developmental pathologies, such as Denys Drash syndrome and preeclampsia. Here, we examine the molecular diversity of VEGF-A as a regulator of its biological activity and compare the role of the pro- and antiangiogenic VEGF-A splice isoforms in both normal and pathophysiological processes.

Keywords: VEGF, splicing, angiogenesis

Neovascularisation, the formation of new blood vessels, is orchestrated via two distinct mechanisms, namely vasculogenesis and angiogenesis. Vasculogenesis, a process of blood-vessel growth that mainly occurs during embryonic development, involves the in situ differentiation of endothelial cell progenitors (angioblasts) from precursor cells (hemangioblasts) [133]. In contrast, angiogenesis is considered to be the development and remodeling of new blood vessels from an already existing vasculature [133]. In the adult, angiogenesis plays an important role in pregnancy, the female menstruation cycle [126,131], and in tissue growth and repair during wound healing [11,116]. Moreover, angiogenesis is a fundamental regulatory process involved in the pathogenesis of several human diseases, including rheumatoid arthritis [36], ocular neovascular disorders, such as age-related macular degeneration, and proliferative retinopathies [32], cardiovascular disease [19], and cancer [83].

The maintenance of vascular homeostasis is dependent upon the balance of pro- and antiangiogenic factors and is tightly regulated by receptor-ligand interactions, intracellular signaling pathways, as well as interactions between cells and the extracellular matrix [19,44,100]. The switch to the angiogenic phenotype is achieved when an abundance of proangiogenic factors shifts the angiogenic response in favor of vessel growth and remodeling [59]. Vascular endothelial growth factor-A (VEGFA, hereafter referred to as VEGF) is the most potent mediator of this neovascularization event in both health and disease [39,103]. The VEGF gene resides on chromosome 6 [167] and is organized as a single gene consisting of eight exons spanning approximately 14 kbp and separated by 7 introns [64]. The VEGFxxx family of isoforms, where xxx refers to the number of amino acids within a given isoform, is formed by differential splicing in exons 6 and 7 and the proximal splice site in exon 8 (termed exon 8a). Differential splicing of full-length VEGF pre-mRNA gives rise to two known families of proteins consisting of multiple isoforms that differ by only six amino acids at their C-terminus (Figure 1) [8,64]. Conventional VEGFxxx is angiogenic, while the VEGFxxxb isoform family is antiangiogenic [171]. The VEGFxxxb family of isoforms is formed by distal splice-site selection 66 bp downstream of the proximal splice site in exon 8 (termed exon 8b; see Figure 1) [6]. This distal splicing event results in an open reading frame of the exact same number of nucleotides as the proximally spliced variants (i.e., proangiogenic isoforms); however, the translated amino-acid sequence is different (Figure 2) [6], which has implications for the biological properties of the protein. The main focus of this review is to describe the molecular diversity of VEGF isoform expression and the role of these opposing isoforms in maintaining vascular integrity in both normal and pathological physiology.

Figure 1
DNA, RNA, and protein products of human vascular endothelial growth factor (VEGF) families. (A) Gene structure of VEGF. The entire gene sequence of VEGF spans 16,272 bp and is located on chromosome 6p12. (B) Alternative splicing of the VEGF gene gives ...
Figure 2
The amino-acid sequence and exon structure of the 3′ end of VEGFxxx and VEGFxxxb. VEGFxxxb differs from VEGFxxx by just six amino acids in the carboxyl terminus. Proximal splice-site selection produces a protein with the proangiogenic sequence ...


VEGF was discovered in the 1980s by three independent research groups and was initially purified from conditioned media of bovine pituitary folliculostellate cells and a variety of tumor cell lines [42,55,96]. Further characterization of this growth factor revealed its potency as an angiogenic factor and mitogen specific for endothelial cells [42,55,96]. A disulfide-linked dimeric glycoprotein that induced increased extravasation of dye from blood vessels was discovered in 1986, termed vascular permeability factor (VPF) [143]; this protein was subsequently found to be structurally identical to VEGF [80].

The biology of the VEGF proteins is complex [39]. VEGF, the focus of this review, is part of a super-family of cysteine knot proteins, which also includes VEGF-B, -C, -D, and placental growth factor (PlGF). VEGF isoforms are essential regulators of angiogenesis and vascular permeability and elicit their intracellular activities via the activation of two receptor tyrosine kinases (RTKs): VEGFR-1 and -2.

VEGFR-1, a 180-kDa high-affinity fms-like tyro-sine kinase-1 (Flt-1) [31], and VEGFR-2, a 200–230-kDa kinase insert domain-containing receptor (KDR) [125,158], are transmembrane glycoproteins consisting of a seven-tandem immunoglobulin (Ig)-like domain, which serves as the extracellular ligand-binding region (the VEGF-binding site has been mapped to domains 2 and 3 [47,145]), a single-transmembrane domain, and a cytoplasmic domain consisting of two tyrosine kinase catalytic domains (Figure 3) [144,158]. Moreover, it has also been reported that a family of cell-surface glycoproteins, particularly neuropilin-1 (Np-1), act as isoform-specific coreceptors for VEGF-A [148]; initial studies on the VEGFxxx isoforms highlighted that isoforms that lack exon 7 do not bind Np-1 [148]. Further, it has now been identified that the basic carboxy-terminal amino acids at the C′terminus of exon 8a are essential for Np-1 binding [78], and isoforms that lack this moiety (e.g., the VEGFxxxb subfamily) are unable to bind NP-1. The interactions of the various members of the VEGF-A family with their respective receptors are outlined in Figure 3. Ligand binding to the extracellular domain of RTKs, such as VEGFR-2, results in a maximal increase of kinase activity following the induction of receptor dimerization and subsequent phosphorylation of tyrosine residues on the intracellular domain of the receptor [67,139]. This event is crucial for the recruitment of additional signaling molecules that contain SH2 (Src homology 2) or PTB (phosphotyrosine binding) domains, which mediate further downstream signaling cascades [67,140]. Further, the association of RTKs with coreceptors, such as NP-1, in the case of VEGFR-2:VEGF165 signaling/interaction, can enhance the functional signal transduction, which mediates diverse cellular responses [67,140].

Figure 3
Both the pro- and antiangiogenic isoforms of VEGF interact with VEGF receptors. VEGFxxx and VEGFxxxb interact with the VEGFR-2 extracellular domain, resulting in differential downstream kinase activation.


The interactions between VEGF and its receptors are essential for many angiogenic processes both in normal physiology and pathological processes; these ligand receptor associations are further complicated, however, by alternative splicing events, which are now known to give rise to at least 14 subtypes of VEGF, namely, VEGF111, VEGF121, VEGF121b, VEGF145, VEGF145b, VEGF148, VEGF162, VEGF165, VEGF165b, VEGF183, VEGF183b, VEGF189, VEGF189b [105], and VEGF206 [1,24,64,95,106]. Following the discovery of the antiangiogenic isoform of VEGF, VEGF165b, and its associated family of isoforms, a further layer of complexity was added to understanding the regulation of VEGF expression in both health and disease.


VEGF121 arises from alternative splicing of exons 6 and 7 and is a weakly acidic polypeptide, since it lacks 15 basic amino acids within the 44 residues encoded by exon 7 [65]. It is a freely diffusible protein that does not bind to heparin and is secreted from the cell, whereby it promotes the proliferation of vascular endothelial cells, macromolecular extravasation, and angiogenesis [27,85]. VEGF121 is present in large quantities in the kidneys and lungs, but only low levels are observed in the heart and brain under normal physiological conditions (for review, see [154]). Recently, abundant levels of VEGF121, compared to other VEGF isoforms, were found in the rabbit anterior cruciate ligament [63]. VEGF121 shows preferential binding to the larger VEGF receptors, VEGFR-1 and VEGFR-2, and was not thought to specifically interact with Np-1 [50,143]; moreover, this isoform, which lacks exon 7, showed much lower receptor-binding affinity than that of VEGF165 [53,157]. Recently, however, it was demonstrated that Np-1 and -2 can enhance VEGF121-stimulated signal transduction via the phosphorylation of VEGFR-2, even though VEGF121 did not appear to bind neuropilins directly [146]. Further studies have demonstrated that blocking the functional Np-1 receptor reduced VEGF121-induced endothelial cell migration and sprout formation [111]. Although it was also shown that VEGF121 binds directly to Np-1, this interaction was not sufficient to create the Np-1/VEGFR-2 complex [111].

The affinity of the VEGF splice variants to bind heparin has an overall effect on isoform spatial distribution; these variations then determine whether blood-vessel growth is organized and directed, or disordered [17,50,136]. For example, when mice were engineered to express only the covalently linked homodimeric protein, VEGF120 (VEGF120/120; mouse VEGF is one amino acid shorter than in humans), they exhibited a significant decrease in capillary branch formation, a disruption of blood-vessel growth, and an impairment of directed extension of endothelial cell filopodia during neural tube development [136]. Moreover, in mice lacking the VEGF164 and VEGF188 isoforms, a disruption in vessel development in the eye was observed manifested as severe defects in retinal vascular outgrowth [152].

It is widely accepted that VEGF is upregulated in many cancers. Specific isoform analysis, using quantitative real-time polymerase chain reaction (PCR), determined the relative amounts of VEGF isoform expression in normal and malignant human and mouse prostate tissue [19]. In normal human prostate tissue, the balance of isoform expression favored the production of VEGF165 over VEGF121; in the malignant form, however, there was a significant shift toward VEGF121 expression. When the relative amount of VEGF121 was increased (using morpholino phosphorodiamide antisense oligonucleotide technology), there was a striking increase in prostate tumor angiogenesis, suggesting an important role for VEGF121 in prostate tumorigenesis [20]. VEGF121 is also upregulated in human colon cancer, where it is hypothesized to play an important role in this proliferative, angiogenic process due to its bioavailability [163]; other splice variants, such as VEGF165, VEGF189, and VEGF145, were also consistently expressed. There is some question, however, over the extent to which heparin binding is essential for the localization of VEGF in adult tissues, as it has been shown that the delivery of VEGF genes encoding these isoforms by myoblast-mediated gene transfer produced similar localized vascular effects, regardless of heparin-binding affinity [151]. Recently, it was found that VEGF121 promotes lymphangiogenesis in the sentinel lymph nodes of non-small-cell lung carcinoma patients, where it is hypothesized that VEGF121 may act, at least in part, via the induction of VEGF-C [77]. In addition, VEGF121 also plays a significant role in attenuating hypertension and improving kidney damage in a rat model of preeclampsia [98].


VEGF165 was the first VEGF splice-variant isoform described [42] and is widely considered to be a potent enhancer of vascular permeability and the predominant regulator of both physiological and pathological angiogenesis. VEGF165 is secreted as a 46-kDa homodimer; however, 50–70% of this isoform remains cell- and extracellular matrix–associated due to moderate heparin-surface glycoprotein (HPSG) interactions [65]. Clearly, the heparin-binding domains encoded by exons 6 and 7 can regulate the bioavailability of VEGF, thereby adding complexity to the control of angiogenesis [64].

VEGF165 binds to VEGFR-1 with high affinity (Kd: 10–20 pM): however, the downstream effect of VEGFR-1 kinase activity is relatively weak [29,70,138,168]; thus, it has been suggested that VEGFR-1 may act as a decoy receptor. There is conflicting evidence concerning the ability of VEGFR-1 to facilitate VEGF165-dependent migration and proliferation of endothelial cells in culture, with some groups finding no effect [66,91,168], while others support a role for VEGFR-1 in VEGF165-mediated mitogenic activity [69] and cellular migration [75]. Although VEGFR-2 has a lower binding affinity for VEGF165 (75 pM), compared to VEGFR-1 [159], the binding of VEGF165 to VEGFR-2 leads to a conformational change in VEGFR-2 [135], resulting in receptor dimerization and autophosphorylation of tyrosine 1173 (Y1175 in humans) that can be readily detected in intact endothelial cells [2,47]. Phosphorylation of tyrosine residues 1054 and 1059 in the kinase domain are required for receptor activation, as they are able to hold open the ATP-binding pocket of the tyrosine kinase [82].

VEGF165 elicits its proliferative effects on endothelial cells via the induction of several signaling pathways, including Ras-independent induction of the Raf-MEK-p44/p42MAPK pathway, through PLC-γ activated PKC [155]. In addition, VEGF165 promotes cell survival via the activation of PI3-kinase and its downstream effector protein, Akt [49,52,123,160,122,162]. It has also been reported that VEGF165 promotes the migration of endothelial cells via the stimulation of focal adhesion kinase (FAK) and the subsequent activation of the PI3-kinase-Akt pathway, possibly through the formation of a FAK-PI3-kinase complex [15,122]. VEGF165 appears to mediate the upregulation of antiapoptotic proteins, such as bcl-2 [49] and the inhibitors of apoptosis proteins (IAP) [162]; these pathways work together to promote an environment that is permissive to the activation of proangiogenic gene expression [90]. The transduction of VEGF165-mediated signals also involves Np-1, which is thought to associate with VEGFR-2 to form a functional signaling complex that can enhance VEGF165 signal transduction [148].

Through its interactions with a number of receptors, VEGF165 induces distinct behavioral responses, such as proliferation, migration, and survival, in endothelial cells and is, therefore, able to regulate most, if not all, of the steps involved in both physiological and pathological angiogenesis.

Indeed, in in vitro models, such as the three-dimensional cell-culture model [113,114], and in vivo models, such as the chick chorioallantoic membrane [96], the mouse matrigel assay [104], and the rabbit corneal eye pocket assays [117], VEGF165 consistently causes an angiogenic response. VEGF165 evokes a strong angiogenic response in a number of physiological processes, including embryonic implantation (reviewed in [87]), the female reproductive cycle (reviewed in [126,131]), and wound healing and tissue repair (reviewed in [18,41,44,170]). Moreover, VEGF165 also functions as a regulator of vascular permeability in vivo, where it plays an important role in initiating angiogenesis [6]. Although VEGF165 primarily targets endothelial cells, several studies have reported its role in the function and maintenance of nonendothelial cells, such as lymphocytes [120], retinal pigmental cells [58], and Schwann cells [149]. Moreover, VEGF165 is a potent autocrine survival factor for podocytes [45], hematopoietic cells [76], and cancer cells [3,118] and is neuroprotective in a murine model of motor neurone disease [89]. Since its discovery, the role of VEGF in the regulation of vascular homeostasis has been extensively studied and has provided insight into the molecular mechanism of angiogenesis [19,41,44]. The VEGF165 splice variant is essential in embryogenic vasculogenesis and angiogenesis [17,40]. Targeted gene knockout mice expressing only VEGF164 are normal, while inactivation of this isoform caused embryonic lethality and embryos showed defective vascularization in retinal, coronary, and renal artery development [40]. Clearly, the various VEGF splice isoforms do not demonstrate equivalent function [175].

In addition to the importance of VEGF165 in normal physiology, there are multiple lines of evidence demonstrating the role of VEGF165 in pathological angiogenesis in diseases as diverse as cancer, rheumatoid arthritis, psoriasis, ocular neovascular disorders, and cardiovascular disease [18,20,33,37]. It is well established that the VEGF165 isoform is directly involved in the mediation of vascularization in diabetic retinopathy and age-related macular degeneration [56]. For example, VEGF165 levels are elevated in the eyes of patients with diabetic retinopathy and the specific inhibition of this splice variant with pegaptanib sodium (Macugen®, Pfizer), an RNA oligonucleotide aptamer that binds and inactivates VEGF165, appears to reverse the blood-retinal barrier breakdown associated with this disease [153]. Ranibizumab (Lucentis, Genentech, San Francisco, USA) [134], an intravitreal anti-VEGF antibody, is used effectively for the treatment of neovascular diseases, such as wet age-related macular degeneration [172]. Moreover, VEGF165 is essential for the growth of the vast majority of tumors [43]. VEGF expression is correlated with tumor progression and poor prognosis in breast cancer [48,137] and non-small-cell lung cancer [51,156,157]. The VEGF165 isoform is specifically upregulated in many cancers, including human colorectal cancer [163] and hepatocellular carcinoma [97]. VEGF165 is, therefore, a potential target in anticancer therapies; phase II and III trials showed significant efficacy of anti-VEGF therapies in colorectal [102] and renal-cell carcinoma [173]. As a result, the first anti-VEGF agent, Avastin (bevacizuumab) (Genentech, San Francisco, USA) has now been licensed for colorectal, breast, and non-small-cell lung cancer by the U.S. Food and Drug Administration (FDA) in the United States [38].


Longer forms of VEGFxxx, containing the amino acids encoded by exons 6 and 7, have been identified; VEGF189 and VEGF206 bind to heparin-containing proteoglycans with high affinities [65]. Although these isoforms are cell associated after secretion and generally retained in the extracellular matrix [64,112], they can be released by heparinases or heparin, or cleaved by plasmin to release a more diffusible form of VEGF, termed VEGF110 [84]. The cleavage of VEGF189 results in a dramatic reduction of mitogenic activity on vascular endothelial cells, compared to the alternative isoform, VEGF165 [40,176]. VEGF189 is a basic polypeptide; it has a 24-amino-acid insertion containing a high proportion of basic residues. The variances in isoelectric focus, as well as its high affinity for heparin, significantly affects the bioavailability of VEGF189, compared to VEGF165 [112]. Although it is widely accepted that VEGF121 and VEGF165 are stronger mitogens [84], VEGF189 may play an important role during phases of lower angiogenic potential, where longer isoforms are available for cleavage by plasmin and subsequent release in the soluble form, VEGF110 [163]. VEGF189 induces endothelial cell proliferation and migration in vitro to a similar extent to that observed with VEGF165; however, this effect is hugely dependent on endothelial cell origin, with significant effects observed in human umbilical endothelial cells (HUVECs) but not dermis-derived endothelial cells [60]. It has also been suggested that VEGF189 plays a role in supporting cell adhesion and survival via interactions with the alpha(v)beta3 integrin [68].

In VEGF188/188 mice expressing only the longer isoform, the phenotype consists of impaired retinal arterial vessel development, dwarfism, disrupted chondrocyte development and epiphyseal vascularization, and knee joint dysplasia [101]. The expression profile of VEGF has been examined in osteoblasts, where it was shown that VEGF isoform expression is regulated in a spatiotemporally mechanical-controlled manner, with VEGF189 preferentially expressed under conditions of increased stretch-induced cell tension, where it is thought to play a role in vascularization during bone repair [35]. VEGF189 is also highly expressed in normal and diabetic cardiac and vascular tissue, albeit to a lesser degree than the more soluble VEGF isoforms, VEGF165 and VEGF121 [177].

VEGF isoforms are known to play pivotal roles in the progression and clinical outcome associated with a variety of cancers. In several studies, the expression of VEGF189, compared with VEGF165, was assessed in patients with pulmonary adenocarcinoma [108] or non-small-cell lung cancer [110,127]. Nishi et al. [108], demonstrated increased expression of VEGF189 in patients with poorer prognosis, including distant metastases and enhanced venous involvement. Oshika et al. [110] showed that VEGF189 levels were closely associated with the progression of disease, whereas Regina et al. [127] demonstrated that VEGF189 levels were 10 times higher than those of VEGF165 and were associated with 12 K-ras mutations. Moreover, it has been demonstrated that high expression levels of VEGF189 correlate with increased intratumoral microvessel count, reduced survival rates, and early postoperative relapse [174]. In contrast, VEGF165 and VEGF206 showed no association with these variables, suggesting that the specific isoform, VEGF189, may be a good prognostic marker for patients with non-small-cell lung cancers [174]. The role of VEGF189 in other cancers is also under investigation. Hervé et al. [61] demonstrated that VEGF189 participates in mammary tumor growth, and their study suggests, for the first time, that this longer VEGF splice variant interacts with Np-1 to evoke an autocrine effect of VEGF189 on breast cancer cells [61]. VEGF189 is also increased in renal-cell carcinoma, where it is associated with tumor progression [71], and in hepatocellular carcinoma, where it may play a role in modulating angiogenesis and carcinogenesis [97]. Clearly, VEGF189 contributes to local blood-vessel growth and tumor development in the cancer microenvironment.


The longer forms of VEGF can be cleaved by plasmin to generate a number of smaller, diffusible fragments of VEGF [84]. One of these framents, VEGF110, was found to be a carboxyl-terminal polypeptide, which bound heparin. VEGF110 was subsequently shown to be resistant to further chemical or proteolytic degradation [81]. The mitogenic activity of this isoform was significantly diminished, compared to VEGF165, and its bioactivity was markedly reduced [84]. The binding affinity of ranibizumab (Lucentis), a humanized antibody fragment directed against specific VEGF isoforms, including VEGF110, has been assessed [99]. Biacore analysis demonstrated that ranibizumab was capable of binding to VEGF165, VEGF121, and VEGF110 and inhibited the biological activity of these VEGF isoforms [99]. Although it has been proposed that VEGF110 is angiogenic, the evidence, to date, suggests that this isoform is, at best, only a modest stimulator of endothelial cell proliferation; further investigation is required to fully elucidate the importance of this factor as an in vivo angiogenic agent.


In 2007, a novel, biologically active form of VEGF, VEGF111, was identified [106]. Expression of VEGF111, which is encoded by exons 1–4 and 8, is induced by ultraviolet B and gentoxic drugs, but is not present in healthy human or murine cells. This isoform is both remarkably resistant to proteolysis and is entirely diffusible; these characteristics, coupled with its mitogenic and chemotactic properties, make VEGF111 a potential target for therapies directed at the treatment of ischemic diseases [106]. The potent angiogenic effects of VEGF111, and its potential candidacy in the treatment of ischemic disease, is currently under investigation.


In 1993, Charnock-Jones et al. identified and localized VEGF145 in human uterus and endometrial carcinoma cell lines [23]. The VEGF145 isoform lacks exons 6b and 7; however, it retains a 72-nucleotide sequence encoding 24 amino acids from exon 6a. Recombinant VEGF145 binds to heparin and has also demonstrated binding affinity for Np-2 [54] and VEGFR-2 on endothelial cells [119]. Although considered to be freely soluble, VEGF145 binds to the basement membrane produced by corneal endothelial cells [119]; this VEGF splice variant induces the proliferation of vascular endothelial cells and promotes an angiogenic response in vivo [119].

Although the biological activity of VEGF145 has yet to be fully elucidated, significant levels of this isoform have been reported in tissues related to the female and male reproductive tracts. High levels of VEGF145 have been described in human placenta and cultured placental fibroblasts [1], as well as in ovine placenta and fetal membranes [24]. Further studies in rat models have found the presence of VEGF145 in both the adult rat lung and penis [13], suggesting that this rare variant may have more diverse functions than previously reported. In 2007, Ribeiro et al. [132] demonstrated the presence of VEGF145 in the swine corpus luteum during late luteal phase, compared with VEGF164 or VEGF120, which showed their highest levels early on, and concluded that the differential regulation of VEGF isoform expression indicated specific physiological roles during the growth and regression of the luteal vascular bed. High levels of VEGF145 have also been reported throughout endometrial carcinoma cell lines and several other tumorigenic cell types originating from the female reproductive tract [119]. Subsequently, VEGF145 expression has been described in solid malignant tissues, such as human colon cancer, where it plays a role in proliferative angiogenesis [163].


VEGF148 mRNA, which lacks exon 6 and the terminal part of exons 7 and 8, was identified in isolated single normal human glomeruli [169]. This truncated splice variant, which results from a 35-bp (base pair) deletion at the end of exon 7, may have no biological activity, since it lacks exon 8; thus, the physiological importance of VEGF148 remains to be determined.


In 2003, Lange et al. [92] described the expression of a novel VEGF isoform in A431 ovarian carcinoma cells, VEGF162, which contains exons 1–5, 6, and 8 of the VEGF gene. It is thought that this isoform may have wrongly been interpreted as VEGF165, given its proximity in size. VEGF162 demonstrated binding to the basement membrane produced by corneal endothelial cells and was found to be biologically active; it induced the proliferation of endothelial cells in vitro and angiogenesis in vivo [92]. The specific biological role of this splice variant, however, has yet to be examined.


VEGF183 was first described in 1998 [95] and was further characterized by Jingjing et al. [74] in 1999. Analysis of the nucleotide sequence of VEGF183 revealed an 18-bp deletion, corresponding to the six amino acids, Tyr-Lys-Ser-Trp-Ser-Val, immediately upstream of the exon 7–encoded sequence in VEGF189 [74]. The protein product is six amino acids shorter than VEGF189 as a consequence of an alternative splicing event within exon 6A [74]. The 18-bp deletion in exon 6A may contribute to a novel functionality associated with this unique VEGF isoform, affecting both cell-surface and extracellular matrix–binding properties, when compared to its sister isoform, VEGF189 [73]. VEGF183 is secreted from endothelial cells at low concentrations, where it is predominantly cell associated. This splice variant does not exhibit mitogenic or proliferative capabilities unless released by heparin or cleaved by plasmin, although it does increase vascular permeability of rat blood vessels [73].

High levels of VEGF183 have been described in human brain, spleen, and heart [95], and retinal-derived tissues [74], as well as in other mammalian species, including rat, mouse, guinea pig, and dog tissues [73], as well as rabbit anterior cruciate ligament; however, in this tissue, there is a striking age-related decrease in polypeptide expression, indicating differential actions during development and aging [63]. VEGF183 expression is upregulated in retinal glial cells in vitro under conditions of hypoxia [74]. Increased levels of VEGF183 have also been reported in the medial and lateral meniscus of rabbits, where it was found that VEGF183 and VEGF189 were upregulated throughout a 24-hour period, whereas the more soluble isoforms peaked at eight hours, suggesting differential function of the various VEGF isoforms [62]. When levels of VEGF isoform expression were studied in human pancreatic carcinoma tissues, it was found that all cell lines expressed both the soluble VEGF isoforms as well as VEGF183 [147]. It may be that the modulation of VEGF183 expression by hypoxia provides a pathway for VEGF induction in a number of vascular tissues in both health and disease; additional studies are required to clarify the role of VEGF183, especially in comparison to VEGF189.


In 1991, a fourth molecular form of VEGF, termed VEGF206, was identified by Houck et al. [64]. This splice variant includes a 41-amino-acid insertion relative to VEGF165 and contains the same highly basic 24-amino acid insertion described in VEGF189 [64]. Sequence analysis of the VEGF gene demonstrated that an alternative splicing event is responsible for the generation of VEGF206 isoform; moreover, although VEGF206 possesses the signal sequence required for secretion, this splice variant is poorly exported from the cell and does not demonstrate mitogenic properties [64]. Similarly to VEGF189, VEGF206 is tightly bound to extracellular heparin-containing proteoglycans [112], which clearly affects the bioavailability of this molecule.

VEGF206 expression was originally thought to be restricted to cells of placental origin, where it increases growth factor levels in the extracellular matrix and affects vascular permeability [2,26]. Subsequently, it was found that VEGF206 is constitutively expressed in secretory granules of isolated human skin mast cells of both normal and leukemic origin [57] and is detected in some non-small-cell lung cancer samples, although it demonstrated no statistical correlation with tumor angiogenesis or overall survival [174].

Clearly, the extracellular matrix functions as a storage depot for various VEGF isoforms, allowing for the slow release of these molecules and a resultant prolonged angiogenic stimulation. The role of these isoforms in the progression of healthy tissues and in pathological conditions remains to be clearly elucidated, and potential therapeutic strategies involving agents that target these isoforms are in their infancy.


As described above, the proangiogenic isoforms of VEGF are overexpressed in almost all cancers investigated, including renal-cell carcinoma [161], ovarian epithelial tumors [150], lung carcinoma [5], and colon carcinoma [5]. The relative expression of the various proangiogenic isoforms is correlated with tumor stage and development. VEGF189 is the dominant isoform in normal lung tissue; however, in lung carcinoma, the expression pattern shifts to favor production of the smaller isoforms, VEGF121 and VEGF165 [25]. Until recently, only proangiogenic isoforms of VEGF have been described, largely as a consequence of PCR, probe, and antibody design issues, which have overlooked the existence of an alternative splice variant of VEGF, termed VEGFxxxb [6].


VEGF165b was first identified in 2002, as an alternative splice isoform of conventional VEGF, generated by differential splice-site selection in the 3′UTR of the VEGF gene (Figure 2) [6]. This alternate splicing event gives rise to two subexons, exon 8a and 8b. Splice-site selection in exon 8b results in an open reading frame of 18 nucleotides. VEGFxxxb isoforms resulting from this C′terminal exon 8 splicing event, generate proteins of the same length as VEGFxxx isoforms; however, the terminal six amino acids are different, since exon 8a codes for Cys-Asp-Lys-Pro-Arg-Arg and exon 8b for Ser-Leu-Thr-Arg-Lys-Asp [6]. This change has profound implications in terms of structure, receptor interaction, signaling, and properties, as described below. There are a number of amino-acid residue changes predicted to profoundly alter the configuration of the protein. In exon 8b, the loss of Cys-160, which forms a disulfide bond with Cys-146 in exon 7 in VEGF165 [26], results in a key alteration in the tertiary structure of VEGF165b (Figure 2). This alteration is compounded by the replacement of highly charged C′terminal arginines in VEGF165 with neutral lysine-aspartic acid in VEGF165b [22] and the substitution of a proline with an arginine residue. These alterations result in the loss of the putative kink present in VEGFxxx molecules (Figure 2) [6]. So far, the expression of VEGF121b, VEGF165b, VEGF145b, and VEGF189b has been identified [115].


VEGF165b was the first member of the VEGFxxxb family to be identified and was discovered in human renal cortex by reverse-transcriptase (RT)-PCR [6]; this antiangiogenic isoform was later reported to be expressed endogenously in differentiated, but not de-differentiated, human podocytes [28]. The development of specific antibodies and probes against the VEGFxxxb splice variants facilitates the selective detection of this family of isoforms at both the protein level by sandwich ELISA using VEGFxxxb antibody as a capture and a pan-VEGF antibody to detect the bound VEGFxxxb (R&D Systems, Minneapolis, USA), immunohistochemistry, and Western blotting (AbCam, Cambridge MA, USA) and also at the RNA level by quantitative polymerase chain reaction (qPCR) (PrimerDesign, Southampton, UK). VEGF165b levels have been found in abundance in several primary cell lines, including differentiated podocytes, retinal pigmented epithelial cells, and colonic epithelial cells [28,164]. The expression levels of VEGF165b have also been examined in a wide range of normal nonangiogenic normal human tissues, in which VEGFxxxb isoforms appear to be highly significant proportions of total VEGF, sometimes outstripping VEGFxxx isoforms (Figure 4). VEGFxxxb isoforms have been detected in the human vitreous fluid [115] and a number of other tissues, including lung, bladder, colon, islets, kidney, smooth muscle, circulating plasma, urine, and placenta, thus confirming that VEGF165b and other members of the VEGFxxxb family can be secreted from cells (Figure 4). Moreover, VEGFxxxb forms significantly more than, or close to, 50% of the total VEGF protein in many of these tissues [9,86,115], although there is a wide degree of variation [5], ranging from 1% in placental tissues [8] to over 95% in normal colon tissue [164]. Recently, it was reported that VEGF165b is expressed in normal nonlactating human and mouse breast and is down-regulated during lactation [124]. A transgenic mouse line expressing VEGF165b under control of the mouse mammary tumor virus promoter shows increased expression of this antiangiogenic isoform during mammary development, resulting in an inhibition of blood-vessel development, impaired alveolar coverage of the fat pad, and a significant reduction in milk production [124]. Moreover, VEGF165b inhibition in the developing ovary results in follicle progression similar to stimulation with angiogenic isoforms, indicating an endogenous role for antiangigoenic isoforms in the regulation of follicle development [2b]. Additionally, inhibition of VEGFxxxb isoforms in developing testes stimulated vascular development and perturbed testicular cord formation, in a similar manner to the addition of excess VEGF164 [30]. Clearly, there is an important role for VEGFxxxb in normal physiology, with its expression dependent on the angiogenicity of the tissue, being downregulated in angiogenic, or potentially angiogenic, tissues such as placenta, benign prostatic hyperplasia respectively [9,86,115], and mammary alveolar development during lactation [124].

Figure 4
VEGFxxxb isoforms form the majority of VEGF in many tissues and are major components of the VEGF expression in all nonangiogenic tissues, so far, examined [9,86,115].

In immortalized cells used in several laboratories (A375 melanoma, LS174t and HT29 colorectal carcinoma, Chinese hamster ovary [CHO] cells, EJ29 bladder cancer cells, and proliferating podocytes [28,164]) VEGFxxxb isoforms form a minority of VEGF. In primary cultured cells, however (e.g., differentiated podocytes, retinal pigmented epithelial [RPE] cells or colonic epithelial cells [28,164]), VEGF165b levels are significant and can predominate.


Unlike other VEGF isoforms studied, VEGF165b has been reported to be downregulated in all cancers investigated so far, including renal-cell [6], prostate [171], and colon carcinoma [164] and malignant melanoma [121]. Moreover, VEGF121b was reported to inhibit endothelial cell migration and tumor growth in colorectal cancer [130], indicating that the entire family is likely to be antiangiogenic. Recently, it was shown at both the protein and mRNA levels that over 90% of normal colonic tissue was VEGFxxxb; however, in colorectal carcinoma samples, an angiogenic “switch” occurred, and the balance shifted to favor expression of the proangiogenic isoforms [164]. In addition, it was demonstrated that VEGFxxxb binds to, and inhibits, bevacizumab, an anti-VEGF-A antibody, suggesting that the effectiveness of bevacizumab treatment in human colorectal carcinoma may depend upon the balance of VEGF isoforms [164].

In addition to its downregulation in cancer, VEGFxxxb isoform expression is also altered in other microvascular diseases characterized by excess neovascularization or microvascular permeability, including proliferative eye disease, preeclampsia, and Denys-Drash syndrome [9,115,141]. In the eye, VEGFxxxb is downregulated in the vitreous fluid of patients with proliferative diabetic retinopathy; in normal vitreous, 65% of VEGF protein is VEGFxxxb; however, in diabetes, this drops to 16% [115]. In a rodent model of glaucoma, however, VEGFxxxb was upregulated [34]. VEGFxxxb is also downregulated in preeclamptic placentae at term, even though total VEGF levels are increased [8], and is switched off in placenta of type 1 diabetic patients, but not in gestational diabetes [93], and its expression is inhibited by insulin perfusion [142]. In addition, plasma VEGF165b levels at the end of the first trimester have been shown to be a better predictor of those patients who will subsequently suffer from preeclampsia than soluble fms-like tyrosine kinase (sFlt) or endoglin [10]. A dysregulation of VEGFxxxb expression is also observed in the glomeruli of humans with Denys-Drash syndrome, a disease in which mutations in Wilms' tumor-1 gene affect the transcriptional regulation of VEGF expression, where VEGF165b was downregulated [141]. Although the mechanisms underlying the regulation of VEGF gene splicing have not been fully elucidated, it has been shown that external stimuli, such as transforming growth factor-beta (TGF-β) can upregulate VEGFxxxb expression, whereas treatment with insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), and tumor necrosis factor-alpha (TNF-α) favor proximal splice-site selection (i.e., splicing in exon 8a producing VEGFxxx isoforms), resulting in a reduction in the VEGFxxxb levels [109].

Inhibition of p38-MAPK and the Clk/sty splicing factor kinase family, but not ERK1/2, prevented the TGF-β-induced distal splice-site selection involving the SRp55 splicing factor, whereas overexpression of the splicing factors, ASF/SF2 and SRp40, both favored the production of proangiogenic VEGF isoforms [109]. It may, therefore, be possible to interfere with this splice-site selection as a potential antiangiogenic strategy.


The unique six-amino-acid sequence encoded by exon 8b at the C′terminus of the protein gives VEGF165b and VEGF121b (and, by extension, other VEGFxxxb isoforms) radically different properties to those of their sister/conventional VEGFxxx isoforms (i.e., VEGF165 and VEGF121). As described above, VEGF165 binding stimulates endothelial cell migration and proliferation in vitro [38], vasodilatation [88], chronically increased vascular permeability [7], angiogenesis [40], and pathological retinal neovascularization in vivo [107]. In an almost entirely opposite manner, VEGF165b inhibits VEGF165-mediated endothelial cell proliferation [6] and migration [6] in vitro and vasodilatation ex vivo [6]. Further, numerous in vivo models have clearly demonstrated that VEGF165b can specifically inhibit VEGF165-induced angioge in mouse [21,124,128,129], rat and rabbit [171], and chick [21], and VEGF165b does not increase chronic microvascular permeability.

Further investigations have extensively studied the overexpression of VEGF165b on tumor growth in colon, prostate, and renal-cell carcinoma and on the growth of Ewing's sarcoma cells and metastatic melanoma in xenografted mouse models, where overexpression of the antiangiogenic isoform was found to inhibit tumor-cell–mediated migration and proliferation of endothelial cells, and the growth of tumors in mice [129,164,171]. Parenteral treatment with VEGF165b can inhibit tumor growth in mice, both primary heterotopic xenografted colon cancers [129] and orthotopic syngeneic disseminated metastatic melanoma in vivo (e.g., Figure 5) [12].

Figure 5
Recombinant human VEGF165b inhibits tumor growth. (A) The effect of increasing doses of VEGF165bon tumor growth of colon carcinoma cells. (B) Dose-response graph showing complete inhibition of tumor growth at 100-μg bi-weekly injection. (C) Metastatic ...


Sequence analysis of VEGF165b revealed that the dimerization and receptor-binding domain present in exons 3 and 4 appeared to be intact; indeed, it is now known that all members of the VEGFxxxb family, so far, identified are capable of homodime rization [171,9,115], and VEGF165b binding to VEGFR-1 has been confirmed [21]. In contrast, it was shown that the inability of VEGF165b to bind the coreceptor, Np-1, and subsequent loss of VEGFR-2/Np-1 complex formation and Y1052 phosphorylation, contributes to the antiangiogenic properties of the factor [79], unlike VEGF isoforms containing the exon 8a sequence (i.e., VEGFxxx), which readily bind Np-1 [72]. Further studies have shown that VEGF165b can displace 125I-VEGF165 from binding to HUVECs and Fc-VEGFR-2 bound to an ELISA plate, with the same affinity as VEGF165 [171]. Although VEGF165b demonstrated equivalent binding to VEGFR-2 as VEGF165, the relative phosphorylation of the receptor was diminished [171]. Thus, it appears that VEGF165b functions as a competitive inhibitor of the major downstream effects of VEGF165. Recently, the mechanisms by which VEGF165b signaling differs from VEGF165 signaling through VEGFR-2 have been partially elucidated. VEGF165 activates its principal signaling receptor, VEGFR-2, by inducing a conformational change when it binds [135], possibly due to the internal rotation of the intracellular domain of VEGFR-2, similar to that reported for the human epidermal growth factor receptor 2 (HER2 receptor) [14]. VEGF165 binding can, therefore, induce tyrosine autophosphorylation by bringing the kinase domain inside the dimer. As described above, Lena Claessen-Walsh's group (Uppsala) [79], have shown that VEGFR-2 is qualitatively differentially phosphorylated by VEGF165b binding; VEGF165b cannot phosphorylate Y1054, the kinase regulatory site, as shown by site-specific phosphoantibody and phosphopeptide mapping experiments. An analogy to the HER2 receptor would indicate that this would be due to a partial intracellular torsional rotation, thus leading to partial activation of the kinase domain. The lack of Y1052 phosphorylation would result in a rapid closure of the ATP-binding site of the kinase and rapid inactivation. This would explain the weak, transient ERK, JNK, and Akt phosphorylation induced by VEGF165b in porcine aortic endothelial (PAE) cells overexpressing VEGFR-2/endothelial cells, when compared to that induced by VEGF165 [171]. Moreover, VEGF165b inhibits VEGF165-mediated proliferation and MAPK phosphorylation through VEGFR2 in astrocytes (and, to a greater extent, in PlGF knockout astrocytes), but interestingly, can stimulate p42/p44 MAPK phosphorylation by itself [46]. It is yet to be determined whether the difference in cellular behavior induced by VEGF165b relative to VEGF165 is due to insufficient downstream signaling or a qualitative alteration in signaling.


Just as VEGF165 can act on nonendothelial cells, as discussed above, VEGF165b also acts as an autocrine or paracrine survival factor in or for such cells. Treatment of epithelial cells such as retinal pigmented epithelial (RPE) cells or podocytes, or colonic adenoma cells with a neutralizing antibody to VEGF165b, results in increased cytotoxicity [9,10]. Conversely, treatment of these cell types with VEGF165b protein reduces cytotoxicity. A recent preliminary report indicated that VEGF165b could also prevent retinal ischemia-mediated neural cell death (Jing Hua, International Symposium on Ocular Pharmacol and Therapeutics, Budapest, 2008). VEGF165b working as a survival factor may explain results whereby glomerular epithelial cell-specific VEGF165 deletion results in podocyte apoptosis and nephropathy [33].


The binding domains that would permit binding to the vast majority of VEGF antibodies, including therapeutic antibodies such as bevacizumab, and most research antibodies are present in VEGF165b. Indeed, binding assays and antibody interaction experiments show that VEGF165b can bind bevacizumab with equal affinity as VEGF165 [164]. Further, VEGF165b expression significantly affects the efficacy of bevacizumab in animal models. In mice bearing VEGF165b-expressing colonic cancer cells, the tumors grow more slowly than in those with VEGF165-expressing cancer cells [164]. The same dose of bevacizumab that reduced tumor growth in VEGF165-expressing tumors did not inhibit tumors expressing VEGF165b in the same model [164]. Combined with the fact that the VEGFxxx:VEGFxxxb ratio can, in many patients, be close to, or less than, 1 [164], these results suggest treatment of these patients with bevacizumab may fail, because VEGF165b will inhibit the effect of this anti-VEGF antibody and vice versa. Studies are ongoing to determine whether assessing the VEGF165:VEGF165b ratio in patients can predicted the responsiveness to bevacizumab. Commercial kits for VEGF165b measurement, such as that recently released by R&D Systems, or standardized immunohistochemical procedures may, therefore, be useful staging tools in the treatment of colorectal cancer. There are limited data on the binding and/or the effect of VEGF165b on other anti-VEGF agents, such as VEGF-TRAP (aflibercept) and VEGF-R TKI, but a preliminary report indicates that pegaptinib, the VEGF aptamer, does not bind VEGF165b. Clearly, further research is required to dissect the effect of VEGF165b on other anti-VEGF agents, but it is possible that non-VEGF binding drugs (e.g., VEGFR-2 TKI) may work in conjunction with VEGF165b, making them more effective.

VEGF appears to be required for normal function. VEGF-R2 inhibitor administration results in normal capillary loss [4], and endothelial-specific VEGF knockout (all isoforms, including VEGF165b) results in adult mortality in mice due to endothelial cell apoptosis and subsequent hemorrhage [94]. As discussed above, VEGF165b acts as a cytoprotective agent for endothelial and epithelial cells [164]. Thus, there are sound reasons to suspect that VEGF165b therapy will be less problematic than agents that target all VEGF isoforms. The most successful antiangiogenic agent, to date, is nonspecific, targeting all VEGF isoforms. VEGFxxx-specific antagonists (e.g., antiexon 8a C′terminus antibodies) may, therefore, be more precise in their targeting of the proangiogenic isoforms.

There is now a growing body of evidence that splicing regulation is a fundamental event in cancer progression [165,166]. VEGF165 and VEGF165b are derived from the same gene, and the control of divergent physiological properties from one gene resides with mRNA splicing, stability, and translation. Initially, transcription and splicing were regarded as separate processes; however, this is no longer thought to be the case. Early in spliceome assembly, the consensus sequences at the 5′ and 3′ sites are recognized by the splicing apparatus (i.e., the splicing choice occurs early in the birth of an RNA molecule). mRNA splicing is mediated by splicing proteins, which form the spliceosome [16]. Splicing is regulated, however, by splicing regulatory factors; thus the cellular processes that control splicing, and the expressed isoforms, may be therapeutic targets.


Collectively, the findings outlined above demonstrate the wide tissue distribution of VEGFxxx and VEGFxxxb in normal and pathological tissues. Although it has yet to be elucidated, it is clear that regulated splicing events are pivotal in determining the angiogenic phenotype of a particular tissue. The discovery of the antiangiogenic family of VEGF splice variants provides an explanation for previous high levels of VEGF being measured in nonangiogenic tissue and the dysregulation of VEGF expression seen in a variety of angiogenic diseases. It may not be the amount of total VEGF protein present, but rather the ratio of VEGFxxxb protein to total VEGF protein that is an important governing factor in determining the relationship between VEGF and angiogenesis in both health and disease.


The authors would like to acknowledge the support of the British Heart Foundation (BS06/005), the Wellcome Trust (058083 and 79633), and the AICR (02-053).


Declaration of interest: The authors report no financial conflicts of interest. The authors alone are responsible for the content and writing of this paper. Steve Harper and David Bates have received financial support for their research, and acted as a consultant for Philogene Inc who hold the license for therapeutic development of VEGF-A165b. Steve Harper and David Bates are inventors on the VEGF-A165b patent David Bates has received travel support from Philogene Inc over the last 12 months.


1. Anthony FW, Wheeler T, Elcock CL, Pickett M, Thomas EJ. Short report: identification of a specific pattern of vascular endothelial growth factor mRNA expression in human placenta and cultured placental fibroblasts. Placenta. 1994;15:557–561. [PubMed]
2. Autiero M, Waltenberger J, Communi D, Kranz A, Moons L, Lambrechts D, et al. Role of PlGF in the intra- and intermolecular cross-talk between the VEGF receptors Flt1 and Flk1. Nat Med. 2003;9:936–943. [PubMed]
2b. Artac RA, McFee RM, Longfellow Smith RA, Baltes-Breitwisch MM, Clopton DT, Cupp AS. Neutralization of vascular endothelial growth factor inhibitory 1 isoforms is more effective than treatment with angiogenic isoforms in stimulating vascular development and follicle progression in the perinatal rat ovary. Biology of Reproduction. 2009 In press. [PMC free article] [PubMed]
3. Bachelder RE, Crago A, Chung J, Wendt MA, Shaw LM, Robinson G, et al. Vascular endothelial growth factor is an autocrine survival factor for neuropilin-expressing breast carcinoma cells. Cancer Res. 2001;61:5736–5740. [PubMed]
4. Baffert F, Le T, Sennino B, Thurston G, Kuo CJ, Hu-Lowe D, et al. Cellular changes in normal blood capillaries undergoing regression after inhibition of VEGF signaling. Am J Physiol Heart Circ Physiol. 2006;290:H547–H559. [PubMed]
5. Bates DO. Molecular diversity of VEGF-A. J Vasc Res. 2008;45:15.
6. Bates DO, Cui TG, Doughty JM, Winkler M, Sugiono M, Shields JD, et al. VEGF165b, an inhibitory splice variant of vascular endothelial growth factor, is downregulated in renal cell carcinoma. Cancer Res. 2002;62:4123–4131. [PubMed]
7. Bates DO, Curry FE. Vascular endothelial growth factor increases hydraulic conductivity of isolated perfused microvessels. Am J Physiol Heart Circ Physiol. 1996;271:H2520–H2528. [PubMed]
8. Bates DO, MacMillan PP, Manjaly JG, Qiu Y, Hudson SJ, Bevan HS, et al. The endogenous antiangiogenic family of splice variants of VEGF, VEGFxxxb, are downregulated in preeclamptic placentae at term. Clin Sci (Lond) 2006;110:575–585. [PubMed]
9. Bevan HS, van den Akker NHS, Qiu Y, Polman JA, Foster RR, Yem J, et al. The alternatively spliced antiangiogenic family of VEGF isoforms, VEGFxxxb, in human kidney development. Nephron Physiol. 2008;110:57–67. [PMC free article] [PubMed]
10. Bills VL, Varet J, Millar AB, Harper SJ, Soothil PW, Bates D. Failure to upregulate VEGF165b in maternal plasma is a first-trimester predictive marker for preeclampsia. Clin Sci (Lond) 2008 In press. [PMC free article] [PubMed]
11. Brown LF, Yeo KT, Berse B, Yeo TK, Senger DR, Dvorak HF, et al. Expression of vascular permeability factor (vascular endothelial growth factor) by epidermal keratinocytes during wound healing. J Exp Med. 1992;176:1375–1379. [PMC free article] [PubMed]
12. Budge JR, Fryer JD, Bates DO. Intraperitoneal administration of recombinant human VEGF165b inhibits dissemination of metatatic melanoma cells in vivo. Microcirculation. 2008;17:18–19.
13. Burchardt T, Burchardt M, Chen MW, Buttyan R, de la Taille A, Shabsigh A, et al. Expression of VEGF splice variants 144/145 and 205/206 in adult male tissues. IUBMB Life. 1999;48:405–408. [PubMed]
14. Burgess AW, Cho HS, Eigenbrot C, Ferguson KM, Garrett TP, Leahy DJ, et al. An open-and-shut case? Recent insights into the activation of EGF/ErbB receptors. Mol Cell. 2003;12:541–552. [PubMed]
15. Byzova TV, Goldman CK, Pampori N, Thomas KA, Bett A, Shattil SJ, et al. A mechanism for modulation of cellular responses to VEGF: activation of the integrins. Mol Cell. 2000;6:851–860. [PubMed]
16. Caceres JF, Kornblihtt AR. Alternative splicing: multiple control mechanisms and involvement in human disease. Trends Genet. 2002;18:186–193. [PubMed]
17. Carmeliet P. Developmental biology. Controlling the cellular brakes. Nature. 1999;401:657–658. [PubMed]
18. Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med. 2000;6:389–395. [PubMed]
19. Carmeliet P. Angiogenesis in health and disease. Nat Med. 2003;9:653–660. [PubMed]
20. Catena R, Muniz-Medina V, Moralejo B, Javierre B, Best CJ, Emmert-Buck MR, et al. Increased expression of VEGF121/VEGF165–189 ratio results in a significant enhancement of human prostate tumor angiogenesis. Int J Cancer. 2007;120:2096–2109. [PubMed]
21. Cebe Suarez S, Pieren M, Cariolato L, Arn S, Hoffmann U, Bogucki A, et al. A VEGF-A splice variant defective for heparan sulfate and neuropilin-1 binding shows attenuated signaling through VEGFR-2. Cell Mol Life Sci. 2006;63:2067–2077. [PubMed]
22. Cebe-Suarez S, Grunewald FS, Jaussi R, Li X, Claesson-Welsh L, Spillmann D, et al. Orf virus VEGF-E NZ2 promotes paracellular NRP-1/VEGFR-2 coreceptor assembly via the peptide RPPR. FASEB J. 2008;22:3078–3086. [PubMed]
23. Charnock-Jones DS, Sharkey AM, Rajput-Williams J, Burch D, Schofield JP, Fountain SA, et al. Identification and localization of alternately spliced mRNAs for vascular endothelial growth factor in human uterus and estrogen regulation in endometrial carcinoma cell lines. Biol Reprod. 1993;48:1120–1128. [PubMed]
24. Cheung CY, Singh M, Ebaugh MJ, Brace RA. Vascular endothelial growth factor gene expression in ovine placenta and fetal membranes. Am J Obstet Gynecol. 1995;173:753–759. [PubMed]
25. Cheung N, Wong MP, Yuen ST, Leung SY, Chung LP. Tissue-specific expression pattern of vascular endothelial growth factor isoforms in the malignant transformation of lung and colon. Hum Pathol. 1998;29:910–914. [PubMed]
26. Claffey KP, Senger DR, Spiegelman BM. Structural requirements for dimerization, glycosylation, secretion, and biological function of VPF/VEGF. Biochim Biophys Acta Prot Struct Mol Enzymol. 1995;1246:1–9. [PubMed]
27. Cohen GB, Ren R, Baltimore D. Modular binding domains in signal transduction proteins. Cell. 1995;80:237–248. [PubMed]
28. Cui TG, Foster RR, Saleem M, Mathieson PW, Gillatt DA, Bates DO, et al. Differentiated human podocytes endogenously express an inhibitory isoform of vascular endothelial growth factor (VEGF165b) mRNA and protein. Am J Physiol Renal Physiol. 2004;286:F767–F773. [PubMed]
29. Cunningham SA, Arrate MP, Brock TA, Waxham MN. Interactions of FLT-1 and KDR with phospholipase C gamma: identification of the phosphotyrosine binding sites. Biochem Biophys Res Commun. 1997;240:635–639. [PubMed]
30. Cupp AS, Bott RC, Pohlmann R, Broeck RAT, Clopton DT. Regulation of vascular endothelial growth factor (VEGF-A) isoforms may be a mechanism to regulate sex-specific vascular development, cord formation, and follicle progression within developing gonads. J Vasc Res. 2008;44:15.
31. de Vries C, Escobedo JA, Ueno H, Houck K, Ferrara N, Williams LT. The Fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science. 1992;255:989–991. [PubMed]
32. Duh E, Aiello LP. Vascular endothelial growth factor and diabetes: the agonist versus antagonist paradox. Diabetes. 1999;48:1899–1906. [PubMed]
33. Eremina V, Sood M, Haigh J, Nagy A, Lajoie G, Ferrara N, et al. Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases. J Clin Invest. 2003;111:707–716. [PMC free article] [PubMed]
34. Ergorul C, Ray A, Huang W, Darland D, Luo ZK, Grosskreutz CL. Levels of vascular endothelial growth factor-A165b (VEGF-A165b are elevated in experimental glaucoma. Mol Vis. 2008;14:1517–1524. [PMC free article] [PubMed]
35. Faure C, Linossier MT, Malaval L, Lafage-Proust MH, Peyroche S, Vico L, et al. Mechanical signals modulated vascular endothelial growth factor-A (VEGF-A) alternative splicing in osteoblastic cells through actin polymerisation. Bone. 2008;42:1092–1101. [PubMed]
36. Fava RA, Olsen NJ, Spencer-Green G, Yeo KT, Yeo TK, Berse B, et al. Vascular permeability factor/endothelial growth factor (VPF/VEGF): accumulation and expression in human synovial fluids and rheumatoid synovial tissue. J Exp Med. 1994;180:341–346. [PMC free article] [PubMed]
37. Favard C, Moukadiri H, Dorey C, Praloran V, Plouet J. Purification and biological properties of vasculotropin, a new angiogenic cytokine. Biol Cell. 1991;73:1–6. [PubMed]
38. Food and Drug Administration (FDA) The FDA approves drugs for colorectal cancer, lung cancer. FDA Consum. 2007:41.
39. Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev. 2004;25:581–611. [PubMed]
40. Ferrara N, Bunting S. Vascular endothelial growth factor, a specific regulator of angiogenesis. Curr Opini Nephrol Hypertens. 1996;5:35–44. [PubMed]
41. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev. 1997;18:4–25. [PubMed]
42. Ferrara N, Henzel WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun. 1989;161:851–858. [PubMed]
43. Folkman J. Role of angiogenesis in tumor growth and metastasis. Semin Oncol. 2002;29:15–18. [PubMed]
44. Folkman J, Shing Y. Angiogenesis. J Biol Chem. 1992;267:10931–10934. [PubMed]
45. Foster RR, Saleem MA, Mathieson PW, Bates DO, Harper SJ. Vascular endothelial growth factor and nephrin interact and reduce apoptosis in human podocytes. Am J Physiol Renal Physiol. 2005;288:F48–F57. [PubMed]
46. Freitas-Andrade M, Carmeliet P, Stanimirovic DB, Moreno M. VEGFR-2-mediated increased proliferation and survival in response to oxygen and glucose deprivation in PlGF knockout astrocytes. J Neurochem. 2008;107:756–767. [PubMed]
47. Fuh G, Li B, Crowley C, Cunningham B, Wells JA. Requirements for binding and signaling of the kinase domain receptor for vascular endothelial growth factor. J Biol Chem. 1998;273:11197–11204. [PubMed]
48. Gasparini G. Prognostic value of vascular endothelial growth factor in breast cancer. Oncologist. 2000;5(Suppl 1):37–44. [PubMed]
49. Gerber HP, Dixit V, Ferrara N. Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells. J Biol Chem. 1998;273:13313–13316. [PubMed]
50. Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, et al. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol. 2003;161:1163–1177. [PMC free article] [PubMed]
51. Giatromanolaki A, Koukourakis MI, Sivridis E, Turley H, Talks K, Pezzella F, et al. Relation of hypoxia inducible factor 1 alpha and 2 alpha in operable non-small-cell lung cancer to angiogenic/molecular profile of tumours and survival. Br J Cancer. 2001;85:881–890. [PMC free article] [PubMed]
52. Gille H, Kowalski J, Li B, LeCouter J, Moffat B, Zioncheck TF, et al. Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants. J Biol Chem. 2001;276:3222–3230. [PubMed]
53. Gitay-Goren H, Soker S, Vlodavsky I, Neufeld G. The binding of vascular endothelial growth factor to its receptors is dependent on cell surface–associated heparin-like molecules. J Biol Chem. 1992;267:6093–6098. [PubMed]
54. Gluzman-Poltorak Z, Cohen T, Herzog Y, Neufeld G. Neuropilin-2 is a receptor for the vascular endothelial growth factor (VEGF) forms VEGF-145 and VEGF-165. J Biol Chem. 2000;275:29922. [PubMed]
55. Gospodarowicz D, Lau K. Pituitary follicular cells secrete both vascular endothelial growth factor and follistatin. Biochem Biophys Res Commun. 1989;165:292–298. [PubMed]
56. Gragoudas ES, Adamis AP, Cunningham ET, Jr, Feinsod M, Guyer DR. Pegaptanib for neovascular age-related macular degeneration. NEJM. 2004;351:2805–2816. [PubMed]
57. Grutzkau A, Kruger-Krasagakes S, Baumeister H, Schwarz C, Kogel H, Welker P, et al. Synthesis, storage, and release of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) by human mast cells: implications for the biological significance of VEGF-206. Mol Biol Cell. 1998;9:875–884. [PMC free article] [PubMed]
58. Guerrin M, Moukadiri H, Chollet P, Moro F, Dutt K, Malecaze F, et al. Vasculotropin/vascular endothelial growth factor is an autocrine growth factor for human retinal pigment epithelial cells cultured in vitro. J Cell Physiol. 1995;164:385–394. [PubMed]
59. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 1996;86:353–364. [PubMed]
60. Herve MA, Buteau-Lozano H, Mourah S, Calvo F, Perrot-Applanat M. VEGF-189 stimulates endothelial cells proliferation and migration in vitro and upregulates the expression of Flk-1/KDR mRNA. Exp Cell Res. 2005;309:24–31. [PubMed]
61. Herve MA, Buteau-Lozano H, Vassy R, Bieche I, Velasco G, Pla M, et al. Overexpression of vascular endothelial growth factor 189 in breast cancer cells leads to delayed tumor uptake with dilated intratumoral vessels. Am J Pathol. 2008;172:167–178. [PMC free article] [PubMed]
62. Hofstaetter JG, Saad FA, Samuel RE, Wunderlich L, Choi YH, Glimcher MJ. Differential expression of VEGF isoforms and receptors in knee joint menisci under systemic hypoxia. Biochem Biophys Res Commun. 2004;324:667–672. [PubMed]
63. Hofstaetter JG, Saad FA, Sunk IG, Bobacz K, Friehs I, Glimcher MJ. Age-dependent expression of VEGF isoforms and receptors in the rabbit anterior cruciate ligament. Biochim Biophys Acta. 2007;1770:997–1002. [PubMed]
64. Houck KA, Ferrara N, Winer J, Cachianes G, Li B, Leung DW. The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA. Mol Endocrinol. 1991;5:1806–1814. [PubMed]
65. Houck KA, Leung DW, Rowland AM, Winer J, Ferrara N. Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms. J Biol Chem. 1992;267:26031–26037. [PubMed]
66. Huang K, Andersson C, Roomans GM, Ito N, Claesson-Welsh L. Signaling properties of VEGF receptor-1 and -2 homo- and heterodimers. Int J Biochem Cell Biol. 2001;33:315–324. [PubMed]
67. Hubbard SR. Structural analysis of receptor tyrosine kinases. Prog Biophys Mol Biol. 1999;71:343–358. [PubMed]
68. Hutchings H, Ortega N, Plouet J. Extra-cellular matrix–bound vascular endothelial growth factor promotes endothelial cell adhesion, migration, and survival through integrin ligation. FASEB J. 2003;17:1520–1522. [PubMed]
69. Ito N, Huang K, Claesson-Welsh L. Signal transduction by VEGF receptor-1 wild type and mutant proteins. Cell Signal. 2001;13:849–854. [PubMed]
70. Ito N, Wernstedt C, Engstrom U, Claesson-Welsh L. Identification of vascular endothelial growth factor receptor-1 tyrosine phosphorylation sites and binding of SH2 domain-containing molecules. J Biol Chem. 1998;273:23410–23418. [PubMed]
71. Jacobsen J, Grankvist K, Rasmuson T, Ljungberg B. Different isoform patterns for vascular endothelial growth factor between clear cell and papillary renal cell carcinoma. BJU Int. 2006;97:1102–1108. [PubMed]
72. Jia H, Bagherzadeh A, Hartzoulakis B, Jarvis A, Lohr M, Shaikh S, et al. Characterization of a bicyclic peptide neuropilin-1 (NP-1) antagonist (EG3287) reveals importance of vascular endothelial growth factor exon 8 for NP-1 binding and role of NP-1 in KDR signaling. J Biol Chem. 2006;281:13493–13502. [PubMed]
73. Jingjing L, Srinivasan B, Roque RS. Ecto-domain shedding of VEGF-183, a novel isoform of vascular endothelial growth factor, promotes its mitogenic activity in vitro. Angiogenesis. 2001;4:103–112. [PubMed]
74. Jingjing L, Xue Y, Agarwal N, Roque RS. Human Müller cells express VEGF-183, a novel spliced variant of vascular endothelial growth factor. Invest Ophthalmol Vis Sci. 1999;40:752–759. [PubMed]
75. Kanno S, Oda N, Abe M, Terai Y, Ito M, Shitara K, et al. Roles of two VEGF receptors, Flt-1 and KDR, in the signal transduction of VEGF effects in human vascular endothelial cells. Oncogene. 2000;19:2138–2146. [PubMed]
76. Katoh O, Tauchi H, Kawaishi K, Kimura A, Satow Y. Expression of the vascular endothelial growth factor (VEGF) receptor gene, KDR, in hematopoietic cells and inhibitory effect of VEGF on apoptotic cell death caused by ionizing radiation. Cancer Res. 1995;55:5687–5692. [PubMed]
77. Kawai H, Minamiya Y, Ito M, Saito H, Ogawa J. VEGF-121 promotes lymphangiogenesis in the sentinel lymph nodes of non-small-cell lung carcinoma patients. Lung Cancer. 2008;59:41–47. [PubMed]
78. Kawamura H, Li X, Goishi K, van Meeteren LA, Jakobsson L, Cebe-Suarez S, et al. Neuropilin-1 in regulation of VEGF-induced activation of p38MAPK and endothelial cell organization. Blood. 2008;112:3638–3649. [PMC free article] [PubMed]
79. Kawamura H, Li X, Harper SJ, Bates DO, Claesson-Welsh L. Vascular endothelial growth factor (VEGF)-A165b is a weak in vitro agonist for VEGF receptor-2 due to lack of coreceptor binding and deficient regulation of kinase activity. Cancer Res. 2008;68:4683–4692. [PubMed]
80. Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J, et al. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science. 1989;246:1309–1312. [PubMed]
81. Keck RG, Berleau L, Harris R, Keyt BA. Disulfide structure of the heparin binding domain in vascular endothelial growth factor: characterization of posttranslational modifications in VEGF. Arch Biochem Biophys. 1997;344:103–113. [PubMed]
82. Kendall RL, Rutledge RZ, Mao X, Tebben AJ, Hungate RW, Thomas KA. Vascular endothelial growth factor receptor KDR tyrosine kinase activity is increased by autophosphorylation of two activation loop tyrosine residues. J Biol Chem. 1999;274:6453–6460. [PubMed]
83. Kerbel RS. Tumor angiogenesis. NEJM. 2008;358:2039–2049. [PubMed]
84. Keyt BA, Berleau LT, Nguyen HV, Chen H, Heinsohn H, Vandlen R, et al. The carboxyl-terminal domain (111–165) of vascular endothelial growth factor is critical for its mitogenic potency. J Biol Chem. 1996;271:7788–7795. [PubMed]
85. Kondo S, Matsumoto T, Yokoyama Y, Ohmori I, Suzuki H. The shortest isoform of human vascular endothelial growth factor/vascular permeability factor (VEGF/VPF121) produced by Saccharomyces cerevisiae promotes both angiogenesis and vascular permeability. Biochim Biophys Acta. 1995;1243:195–202. [PubMed]
86. Konopatskaya O, Churchill AJ, Harper SJ, Bates DO, Gardiner TA. VEGF165b, an endogenous C-terminal splice variant of VEGF, inhibits retinal neovascularization in mice. Mol Vis. 2006;12:626–632. [PubMed]
87. Krussel JS, Bielfeld P, Polan ML, Simon C. Regulation of embryonic implantation. Eur J Obstet Gynecol Reprod Biol. 2003;110(Suppl 1):S2–S9. [PubMed]
88. Ku DD, Zaleski JK, Liu S, Brock TA. Vascular endothelial growth factor induces EDRF-dependent relaxation in coronary arteries. Am J Physiol Heart Circ Physiol. 1993;265:H586–H592. [PubMed]
89. Lambrechts D, Storkebaum E, Morimoto M, Del-Favero J, Desmet F, Marklund SL, et al. VEGF is a modifier of amyotrophic lateral sclerosis in mice and humans and protects motoneurons against ischemic death. Nat Genet. 2003;34:383–394. [PubMed]
90. Lamoreaux WJ, Fitzgerald M, Reiner A, Hasty KA, Charles ST. Vascular endothelial growth factor increases release of gelatinase A and decreases release of tissue inhibitor of metalloproteinases by microvascular endothelial cells in vitro. Microvasc Res. 1998;55:29–42. [PubMed]
91. Landgren E, Schiller P, Cao Y, Claesson-Welsh L. Placenta growth factor stimulates MAP kinase and mitogenicity but not phospholipase C-gamma and migration of endothelial cells expressing Flt 1. Oncogene. 1998;16:359–367. [PubMed]
92. Lange T, Guttmann-Raviv N, Baruch L, Machluf M, Neufeld G. VEGF-162, a new heparin-binding vascular endothelial growth factor splice form that is expressed in transformed human cells. J Biol Chem. 2003;278:17164–17169. [PubMed]
93. Leach L, Reeve KS, Garrioch MA, Bates DO. Effects of gestational diabetes (GDM) on vascular integrity of the human placenta: the role of VEGF165b. Microcirculation. 2006;13:526.
94. Lee S, Chen TT, Barber CL, Jordan MC, Murdock J, Desai S, et al. Autocrine VEGF signaling is required for vascular homeostasis. Cell. 2007;130:691–703. [PMC free article] [PubMed]
95. Lei J, Jiang A, Pei D. Identification and characterization of a new splicing variant of vascular endothelial growth factor: VEGF-183. Biochim Biophys Acta. 1998;1443:400–406. [PubMed]
96. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science. 1989;246:1306–1309. [PubMed]
97. Li Q, Xu B, Fu L, Hao XS. Correlation of four vascular specific growth factors with carcinogenesis and portal vein tumor thrombus formation in human hepatocellular carcinoma. J Exp Clin Cancer Res. 2006;25:403–409. [PubMed]
98. Li Z, Zhang Y, Ying Ma J, Kapoun AM, Shao Q, Kerr I, Lam A, et al. Recombinant vascular endothelial growth factor 121 attenuates hypertension and improves kidney damage in a rat model of preeclampsia. Hypertension. 2007;50:686–692. [PubMed]
99. Lowe J, Araujo J, Yang J, Reich M, Oldendorp A, Shiu V, et al. Ranibizumab inhibits multiple forms of biologically active vascular endothelial growth factor in vitro and in vivo. Exp Eye Res. 2007;85:425–430. [PubMed]
100. Madri JA, Graesser D, Haas T. The roles of adhesion molecules and proteinases in lymphocyte transendothelial migration. Biochem Cell Biol. 1996;74:749–757. [PubMed]
101. Maes C, Stockmans I, Moermans K, Van Looveren R, Smets N, Carmeliet P, et al. Soluble VEGF isoforms are essential for establishing epiphyseal vascularization and regulating chondrocyte development and survival. J Clin Invest. 2004;113:188–199. [PMC free article] [PubMed]
102. McCarthy M. Antiangiogenesis drug promising for metastatic colorectal cancer. Lancet. 2003;361:1959. [PubMed]
103. McColl BK, Stacker SA, Achen MG. Molecular regulation of the VEGF family—inducers of angiogenesis and lymphangiogenesis. APMIS. 2004;112:463–380. [PubMed]
104. Mesri EA, Federoff HJ, Brownlee M. Expression of vascular endothelial growth factor from a defective herpes simplex virus type 1 amplicon vector induces angiogenesis in mice. Circ Res. 1995;76:161–167. [PubMed]
105. Miller-Kasprzak E, Jagodzinski PP. 5-aza-2-deoxycytidine increases the expression of antiangiogenic vascular endothelial growth factor 189b variant in human lung microvascular endothelial cells. Biomed Pharmacother. 2008;62:158–163. [PubMed]
106. Mineur P, Colige AC, Deroanne CF, Dubail J, Kesteloot F, Habraken Y, et al. Newly identified biologically active and proteolysis-resistant VEGF-A isoform VEGF-111 is induced by genotoxic agents. J Cell Biol. 2007;179:1261–1273. [PMC free article] [PubMed]
107. Mitchell CA, Rutland CS, Walker M, Nasir M, Foss AJ, Stewart C, et al. Unique vascular phenotypes following overexpression of individual VEGF-A isoforms from the developing lens. Angiogenesis. 2006;9:209–224. [PubMed]
108. Nishi M, Abe Y, Tomii Y, Tsukamoto H, Kijima H, Yamazaki H, et al. Cell-binding isoforms of vascular endothelial growth factor-A (VEGF-189) contribute to blood flow–distant metastasis of pulmonary adenocarcinoma. Int J Oncol. 2005;26:1517–1524. [PubMed]
109. Nowak DG, Woolard J, Amin EM, Konopatskaya O, Saleem MA, Churchill AJ, et al. Expression of pro- and antiangiogenic isoforms of VEGF is differentially regulated by splicing and growth factors. J Cell Sci. 2008;121:3487–3495. [PMC free article] [PubMed]
110. Oshika Y, Nakamura M, Tokunaga T, Ozeki Y, Fukushima Y, Hatanaka H, et al. Expression of cell-associated isoform of vascular endothelial growth factor 189 and its prognostic relevance in non-small-cell lung cancer. Int J Oncol. 1998;12:541–544. [PubMed]
111. Pan Q, Chathery Y, Wu Y, Rathore N, Tong RK, Peale F, et al. Neuropilin-1 binds to VEGF-121 and regulates endothelial cell migration and sprouting. J Biol Chem. 2007;282:24049–24056. [PubMed]
112. Park JE, Keller GA, Ferrara N. The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix–bound VEGF. Mol Biol Cell. 1993;4:1317–1326. [PMC free article] [PubMed]
113. Pepper MS, Ferrara N, Orci L, Montesano R. Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro. Biochem Biophys Res Commun. 1992;189:824–831. [PubMed]
114. Pepper MS, Wasi S, Ferrara N, Orci L, Montesano R. In vitro angiogenic and proteolytic properties of bovine lymphatic endothelial cells. Exp Cell Res. 1994;210:298–305. [PubMed]
115. Perrin RM, Konopatskaya O, Qiu Y, Harper S, Bates DO, Churchill AJ. Diabetic retinopathy is associated with a switch in splicing from anti- to proangiogenic isoforms of vascular endothelial growth factor. Diabetologia. 2005;48:2422–2427. [PubMed]
116. Peters KG, De Vries C, Williams LT. Vascular endothelial growth factor receptor expression during embryogenesis and tissue repair suggests a role in endothelial differentiation and blood vessel growth. Proc Natl Acad Sci U S A. 1993;90:8915–8919. [PMC free article] [PubMed]
117. Phillips GD, Stone AM, Jones BD, Schultz JC, Whitehead RA, Knighton DR. Vascular endothelial growth factor (rhVEGF-165) stimulates direct angiogenesis in the rabbit cornea. In Vivo. 1994;8:961–965. [PubMed]
118. Pidgeon GP, Barr MP, Harmey JH, Foley DA, Bouchier-Hayes DJ. Vascular endothelial growth factor (VEGF) upregulates Bcl-2 and inhibits apoptosis in human and murine mammary adenocarcinoma cells. Br J Cancer. 2001;85:273–278. [PMC free article] [PubMed]
119. Poltorak Z, Cohen T, Sivan R, Kandelis Y, Spira G, Vlodavsky I, et al. VEGF-145, a secreted vascular endothelial growth factor isoform that binds to extracellular matrix. J Biol Chem. 1997;272:7151–7158. [PubMed]
120. Praloran V, Mirshahi S, Favard C, Moukadiri H, Plouet J. Mitogenic activity of vasculotropin for peripheral human lymphocytes] C R Acad Sci III. 1991;313:21–26. [PubMed]
121. Pritchard-Jones RO, Dunn DB, Qiu Y, Varey AH, Orlando A, Rigby H, et al. Expression of VEGF(xxx)b, the inhibitory isoforms of VEGF, in malignant melanoma. Br J Cancer. 2007;97:223–230. [PMC free article] [PubMed]
122. Qi JH, Claesson-Welsh L. VEGF-induced activation of phosphoinositide 3-kinase is dependent on focal adhesion kinase. Exp Cell Res. 2001;263:173–182. [PubMed]
123. Qi JH, Matsumoto T, Huang K, Olausson K, Christofferson R, Claesson-Welsh L. Phosphoinositide 3 kinase is critical for survival, mitogenesis, and migration but not for differentiation of endothelial cells. Angiogenesis. 1999;3:371–380. [PubMed]
124. Qiu Y, Bevan H, Weeraperuma S, Wratting D, Murphy D, Neal CR, et al. Mammary alveolar development during lactation is inhibited by the endogenous antiangiogenic growth factor isoform, VEGF165b. Faseb J. 2008;22:1104–1112. [PubMed]
125. Quinn TP, Peters KG, De Vries C, Ferrara N, Williams LT. Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium. Proc Natl Acad Sci U S A. 1993;90:7533–7537. [PMC free article] [PubMed]
126. Redmer DA, Reynolds LP. Angiogenesis in the ovary. Rev Reprod. 1996;1:182–192. [PubMed]
127. Regina S, Rollin J, Blechet C, Iochmann S, Reverdiau P, Gruel Y. Tissue factor expression in non-small-cell lung cancer: relationship with vascular endothelial growth factor expression, microvascular density, and K-ras mutation. J Thorac Oncol. 2008;3:689–697. [PubMed]
128. Rennel ES, H-Zadeh MA, Wheatley E, Schuler Y, Kelly SP, Cebe Suarez S, et al. Recombinant human VEGF165b protein is an effective anticancer agent in mice. Eur J Cancer. 2008;44:1883–1894. [PMC free article] [PubMed]
129. Rennel ES, Hamdollah-Zadeh MA, Wheatley ER, Magnussen A, Schuler Y, Kelly SP, et al. Recombinant human VEGF165b protein is an effective anticancer agent in mice. Eur J Cancer. 2008;44:1883–1894. [PMC free article] [PubMed]
130. Rennel ES, Varey AH, Churchill AJ, Harper SJ, Bates DO. VEGF121b is antiangiogenic in cancer and eye disease. Microcirculation. 2008;15:638–639.
131. Reynolds LP, Redmer DA. Expression of the angiogenic factors, basic fibroblast growth factor, and vascular endothelial growth factor, in the ovary. J Anim Sci. 1998;76:1671–1681. [PubMed]
132. Ribeiro LA, Bacci ML, Seren E, Tamanini C, Forni M. Characterization and differential expression of vascular endothelial growth factor isoforms and receptors in swine corpus luteum throughout estrous cycle. Mol Reprod Dev. 2007;74:163–171. [PubMed]
133. Risau W, Flamme I. Vasculogenesis. Annu Rev Cell Dev Biol. 1995;11:73–91. [PubMed]
134. Rosenfeld PJ, Brown DM, Heier JS, Boyer DS, Kaiser PK, Chung CY, et al. Ranibizumab for neovascular age-related macular degeneration. NEJM. 2006;355:1419–1431. [PubMed]
135. Ruch C, Skiniotis G, Steinmetz MO, Walz T, Ballmer-Hofer K. Structure of a VEGF-VEGF receptor complex determined by electron microscopy. Nat Struct Mol Biol. 2007;14:249–250. [PubMed]
136. Ruhrberg C, Gerhardt H, Golding M, Watson R, Ioannidou S, Fujisawa H, et al. Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis. Genes Dev. 2002;16:2684–2698. [PMC free article] [PubMed]
137. Salven P, Perhoniemi V, Tykka H, Maenpaa H, Joensuu H. Serum VEGF levels in women with a benign breast tumor or breast cancer. Breast Cancer Res Treat. 1999;53:161–166. [PubMed]
138. Sawano A, Takahashi T, Yamaguchi S, Shibuya M. The phosphorylated 1169-tyrosine containing region of Flt-1 kinase (VEGFR-1) is a major binding site for PLC-gamma. Biochem Biophys Res Commun. 1997;238:487–491. [PubMed]
139. Schlessinger J. New roles for Src kinases in control of cell survival and angiogenesis. Cell. 2000;100:293–296. [PubMed]
140. Schlessinger J, Lemmon MA. SH2 and PTB domains in tyrosine kinase signaling. Sci STKE. :R. 2003;2003:E12. [PubMed]
141. Schumacher VA, Jeruschke S, Eitner F, Becker JU, Pitschke G, Ince Y, et al. Impaired glomerular maturation and lack of VEGF165b in Denys-Drash syndrome. J Am Soc Nephrol. 2007;18:719–729. [PubMed]
142. Sciota F, Lucas J, Thomas R, Leach L. Insulin perfusion decreases expression of the splice variant VEGF165b in large vessels of the human term chorionic stem villi but not in microvessels. J Vasc Res. 2008;44:87.
143. Senger DR, Perruzzi CA, Feder J, Dvorak HF. A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines. Cancer Res. 1986;46:5629–5632. [PubMed]
144. Shibuya M. Role of VEGF-FLT receptor system in normal and tumor angiogenesis. Adv Cancer Res. 1995;67:281–316. [PubMed]
145. Shinkai A, Ito M, Anazawa H, Yamaguchi S, Shitara K, Shibuya M. Mapping of the sites involved in ligand association and dissociation at the extracellular domain of the kinase insert domain-containing receptor for vascular endothelial growth factor. J Biol Chem. 1998;273:31283–31288. [PubMed]
146. Shraga-Heled N, Kessler O, Prahst C, Kroll J, Augustin H, Neufeld G. Neuropilin-1 and neuropilin-2 enhance VEGF-121 stimulated signal transduction by the VEGFR-2 receptor. FASEB J. 2007;21:915–926. [PubMed]
147. Sipos B, Weber D, Ungefroren H, Kalthoff H, Zuhlsdorff A, Luther C, et al. Vascular endothelial growth factor mediated angiogenic potential of pancreatic ductal carcinomas enhanced by hypoxia: an in vitro and in vivo study. Int J Cancer. 2002;102:592–600. [PubMed]
148. Soker S, Takashima S, Miao HQ, Neufeld G, Klagsbrun M. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell. 1998;92:735–745. [PubMed]
149. Sondell M, Lundborg G, Kanje M. Vascular endothelial growth factor has neurotrophic activity and stimulates axonal outgrowth, enhancing cell survival and Schwann cell proliferation in the peripheral nervous system. J Neurosci. 1999;19:5731–5740. [PubMed]
150. Sowter HM, Corps AN, Evans AL, Clark DE, Charnock-Jones DS, Smith SK. Expression and localization of the vascular endothelial growth factor family in ovarian epithelial tumors. Lab Invest. 1997;77:607–614. [PubMed]
151. Springer ML, Banfi A, Ye J, von Degenfeld G, Kraft PE, Saini SA, et al. Localization of vascular response to VEGF is not dependent on heparin binding. FASEB J. 2007;21:2074–2085. [PubMed]
152. Stalmans I, Ng YS, Rohan R, Fruttiger M, Bouche A, Yuce A, et al. Arteriolar and venular patterning in retinas of mice selectively expressing VEGF isoforms. J Clin Invest. 2002;109:327–336. [PMC free article] [PubMed]
153. Starita C, Patel M, Katz B, Adamis AP. Vascular endothelial growth factor and the potential therapeutic use of pegaptanib (Macugen) in diabetic retinopathy. Dev Ophthalmol. 2007;39:122–148. [PubMed]
154. Takahashi H, Shibuya M. The vascular endothelial growth factor (VEGF)/VEGF receptor system and its role under physiological and pathological conditions. Clin Sci (Lond) 2005;109:227–241. [PubMed]
155. Takahashi T, Ueno H, Shibuya M. VEGF activates protein kinase C–dependent, but Ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells. Oncogene. 1999;18:2221–2230. [PubMed]
156. Tamura M, Ohta Y, Kajita T, Kimura K, Go T, Oda M, et al. Plasma VEGF concentration can predict the tumor angiogenic capacity in non-small-cell lung cancer. Oncol Rep. 2001;8:1097–1102. [PubMed]
157. Tas F, Duranyildiz D, Oguz H, Camlica H, Yasasever V, Topuz E. Serum vascular endothelial growth factor (VEGF) and Bcl-2 levels in advanced-stage non-small-cell lung cancer. Cancer Invest. 2006;24:576–580. [PubMed]
158. Terman BI, Carrion ME, Kovacs E, Rasmussen BA, Eddy RL, Shows TB. Identification of a new endothelial cell growth factor receptor tyrosine kinase. Oncogene. 1991;6:1677–1683. [PubMed]
159. Terman BI, Dougher-Vermazen M, Carrion ME, Dimitrov D, Armellino DC, Gospodarowicz D, et al. Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor. Biochem Biophys Res Commun. 1992;187:1579–1586. [PubMed]
160. Thakker GD, Hajjar DP, Muller WA, Rosengart TK. The role of phosphatidylinositol 3-kinase in vascular endothelial growth factor signaling. J Biol Chem. 1999;274:10002–10007. [PubMed]
161. Tomisawa M, Tokunaga T, Oshika Y, Tsuchida T, Fukushima Y, Sato H, et al. Expression pattern of vascular endothelial growth factor isoform is closely correlated with tumour stage and vascularisation in renal cell carcinoma. Eur J Cancer. 1999;35:133–137. [PubMed]
162. Tran J, Rak J, Sheehan C, Saibil SD, LaCasse E, Korneluk RG, et al. Marked induction of the IAP family antiapoptotic proteins survivin and XIAP by VEGF in vascular endothelial cells. Biochem Biophys Res Commun. 1999;264:781–788. [PubMed]
163. Uthoff SM, Duchrow M, Schmidt MH, Broll R, Bruch HP, Strik MW, et al. VEGF isoforms and mutations in human colorectal cancer. Int J Cancer. 2002;101:32–36. [PubMed]
164. Varey AH, Rennel ES, Qiu Y, Bevan HS, Perrin RM, Raffy S, et al. VEGF165b, an antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: balance of pro- and antiangiogenic VEGF-A isoforms has implications for therapy. Br J Cancer. 2008;98:1366–1379. [PMC free article] [PubMed]
165. Venables JP. Aberrant and alternative splicing in cancer. Cancer Res. 2004;64:7647–7654. [PubMed]
166. Venables JP. Unbalanced alternative splicing and its significance in cancer. Bioessays. 2006;28:378–386. [PubMed]
167. Vincenti V, Cassano C, Rocchi M, Persico G. Assignment of the vascular endothelial growth factor gene to human chromosome 6p21.3. Circulation. 1996;93:1493–1495. [PubMed]
168. Waltenberger J, Claesson-Welsh L, Siegbahn A, Shibuya M, Heldin CH. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. J Biol Chem. 1994;269:26988–26995. [PubMed]
169. Whittle C, Gillespie K, Harrison R, Mathieson PW, Harper SJ. Heterogeneous vascular endothelial growth factor (VEGF) isoform mRNA and receptor mRNA expression in human glomeruli, and the identification of VEGF-148 mRNA, a novel truncated splice variant. Clin Sci (Colch) 1999;97:303–312. [PubMed]
170. Witmer AN, Vrensen GF, Van Noorden CJ, Schlingemann RO. Vascular endothelial growth factors and angiogenesis in eye disease. Prog Retin Eye Res. 2003;22:1–29. [PubMed]
171. Woolard J, Wang WY, Bevan HS, Qiu Y, Morbidelli L, Pritchard-Jones RO, et al. VEGF165b, an inhibitory vascular endothelial growth factor splice variant: mechanism of action, in vivo effect on angiogenesis and endogenous protein expression. Cancer Res. 2004;64:7822–7835. [PubMed]
172. Wylegala E, Teper SJ. [VEGF in age-related macular degeneration. Part II. VEGF inhibitors use in age-related macular degeneration treatment] Klin Oczna. 2007;109:97–100. [PubMed]
173. Yang CC, Chu KC, Yeh WM. Expression of vascular endothelial growth factor in renal cell carcinoma is correlated with cancer advancement. J Clin Lab Anal. 2003;17:85–89. [PubMed]
174. Yuan A, Yu CJ, Kuo SH, Chen WJ, Lin FY, Luh KT, et al. Vascular endothelial growth factor 189 mRNA isoform expression specifically correlates with tumor angiogenesis, patient survival, and postoperative relapse in non-small-cell lung cancer. J Clin Oncol. 2001;19:432–441. [PubMed]
175. Zacchigna S, Pattarini L, Zentilin L, Moimas S, Carrer A, Sinigaglia M, et al. Bone marrow cells recruited through the neuropilin-1 receptor promote arterial formation at the sites of adult neoangiogenesis in mice. J Clin Invest. 2008;118:2062–2075. [PMC free article] [PubMed]
176. Zhang HT, Scott PA, Morbidelli L, Peak S, Moore J, Turley H, et al. The 121-amino-acid isoform of vascular endothelial growth factor is more strongly tumorigenic than other splice variants in vivo. Br J Cancer. 2000;83:63–68. [PMC free article] [PubMed]
177. Zygalaki E, Kaklamanis L, Nikolaou NI, Kyrzopoulos S, Houri M, Kyriakides Z, et al. Expression profile of total VEGF, VEGF splice variants, and VEGF receptors in the myocardium and arterial vasculature of diabetic and nondiabetic patients with coronary artery disease. Clin Biochem. 2008;41:82–87. [PubMed]
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