Molecular Pharmacology of VEGF-A Isoforms: Binding and Signalling at VEGFR2

Abstract

Vascular endothelial growth factor-A (VEGF-A) is a key mediator of angiogenesis, signalling via the class IV tyrosine kinase receptor family of VEGF Receptors (VEGFRs). Although VEGF-A ligands bind to both VEGFR1 and VEGFR2, they primarily signal via VEGFR2 leading to endothelial cell proliferation, survival, migration and vascular permeability. Distinct VEGF-A isoforms result from alternative splicing of the Vegfa gene at exon 8, resulting in VEGF_xxxa or VEGF_xxxb isoforms. Alternative splicing events at exons 5–7, in addition to recently identified posttranslational read-through events, produce VEGF-A isoforms that differ in their bioavailability and interaction with the co-receptor Neuropilin-1. This review explores the molecular pharmacology of VEGF-A isoforms at VEGFR2 in respect to ligand binding and downstream signalling. To understand how VEGF-A isoforms have distinct signalling despite similar affinities for VEGFR2, this review re-evaluates the typical classification of these isoforms relative to the prototypical, “pro-angiogenic” VEGF₁₆₅a. We also examine the molecular mechanisms underpinning the regulation of VEGF-A isoform signalling and the importance of interactions with other membrane and extracellular matrix proteins. As approved therapeutics targeting the VEGF-A/VEGFR signalling axis largely lack long-term efficacy, understanding these isoform-specific mechanisms could aid future drug discovery efforts targeting VEGF receptor pharmacology.

1. Introduction

Angiogenesis is the formation of new blood vessels by the sprouting of endothelial cells from pre-existing vasculature [1]. While vasculogenesis (formation of angioblast-derived blood vessels) occurs mainly during embryonic development, angiogenesis takes place throughout adult life [2,3], playing a vital role in physiological events such as wound repair [4], the oestrous cycle and placentation [5]. In addition to its importance in normal physiology, angiogenesis is also a key feature associated not only with cancer [6], but also with several pathological conditions including age-related macular degeneration [7], rheumatoid arthritis [8], psoriasis [9], diabetes-induced ocular neovascularisation [10], inflammatory diseases [11], ischaemia/reperfusion injury [12], infantile haemangioma and atherosclerosis [13,14]. Despite their distinct aetiologies, these disorders can be characterised as angiogenesis-dependent diseases [15] and are either caused or exacerbated by an imbalance between the production/activity of anti- and pro-angiogenic factors [16]. Among the pro-angiogenic endogenous molecules, vascular endothelial growth factor (VEGF) is a major regulator of blood vessel formation in health and disease [1]. Initially, VEGF was termed vascular permeability factor (VPF) due to its ability to increase the permeability of blood vessels [17,18]. Following subsequent observations of the additional effects of VPF on endothelial cells and concomitant cloning, VPF was renamed VEGF [19].

The VEGF family of proteins comprises VEGF-A, VEGF-B [20], VEGF-C, VEGF-D, Placental Growth Factor (PlGF) [21], the virus-encoded VEGF-E and the snake venom-derived VEGF-F [22,23,24]. VEGF-A is the best characterised family member being the most potent stimulator of angiogenic processes and therefore a target of numerous anti-cancer therapeutics [25]. VEGF-A is a large anti-parallel homodimeric peptide that belongs to the “Cys-loop” superfamily of proteins, based on a central knot motif of cysteine residues that form intramolecular disulphide bonds when assembled into a folded structure [24,26]. VEGF-A is secreted by many cell types such as endothelial cells [27,28], fibroblasts [29], smooth muscle cells [30], platelets [31], neutrophils [32], macrophages and approximately 60% of all tumours [33]. VEGF-A secretion is also induced by ischemia and inflammatory stimuli [34]. Cellular responses to VEGF-A are mainly driven by their binding to their cognate receptor—the vascular endothelial growth factor receptors (VEGFRs). VEGFRs belong to the class IV receptor tyrosine kinase (RTK) family [35] and show similarities to type III RTKs platelet derived growth factor receptor (PDGFR), macrophage colony stimulating factor receptor (M-CSFR), c-KIT and fms-like tyrosine kinase 3 (FLT3) [36]. There are three VEGFR subtypes which are encoded by separate genes: VEGFR1 (Flt-1 in mice) and VEGFR2 (Flk-1; KDR) are structurally similar, whereas VEGFR3 (Flt-4) has a proteolytically processed extracellular domain [37,38]. VEGFRs are expressed by endothelial cells, macrophages, hematopoietic cells and smooth muscle cells [39,40,41]. Signalling of VEGF-A isoforms via VEGFR1 and VEGFR2 drive physiological and pathophysiological angiogenesis, whereas lymphangiogenesis is mediated by VEGF-C/D isoforms via VEGFR3 [42]. Although VEGFR1 has a higher affinity for VEGF-A than VEGFR2, it shows decreased tyrosine kinase activity and is therefore largely considered a decoy receptor that can negatively modulate VEGFR2 activity [43,44]. While VEGFR1 also plays a role in immune cell differentiation [45,46], VEGFR1 is beyond the scope of this review (reviewed in [47]).

Alternative splicing of the Vegfa gene leads to different VEGF-A isoforms which have been proposed to promote distinct signalling outcomes [16]. Through quantifying and comparing the pharmacology of VEGF-A isoforms at VEGFR2, we can begin to comprehend how they differ as distinct endogenous ligands. This could ultimately enable better understanding of molecular mechanisms that give rise to distinct physiological outcomes with relevance in health and disease [16] and future drug discovery efforts [25]. In this review, we have explored in detail the molecular pharmacology of VEGF-A isoforms in terms of their receptor binding to VEGFR2 and downstream signalling with particular reference to the influence of agonist efficacy and signalling coupling on physiological outcomes.

2. Generation of VEGF-A Isoforms by Alternative Splicing

The regulation of vascular supply relies on tight regulation between factors that promote (pro-angiogenic) or inhibit (anti-angiogenic) vessel development, via a mechanism that is reactive to changes in oxygen and nutrient levels. VEGF-A transcription is affected by the local cellular environment, such as during hypoxia [48,49] following secretion of growth factors, cytokines and hormones, shear stress, genotoxic agents [50] and the activity of both oncogenes and tumour suppressor genes [51]. The human Vegfa gene is located on chromosome 6p21.1 [52], with a coding region spanning approximately 14 kilobases consisting of eight exons and seven introns. Alternative splicing of this pre-mRNA selectively removes intron regions and joins specific combinations of exons to generate distinct VEGF-A isoforms [53] (Figure 1). Alternative splicing is advantageous in expanding the repertoire of possible VEGF-A isoforms that can be produced from a single gene [54]. These isoforms differ in respect to their length and are designated VEGF_xxx, where xxx represents the number of amino acids present in the final protein sequence. To date 16 distinct VEGFA isoforms have been identified most commonly from six transcripts: VEGF₁₁₁, VEGF₁₂₁, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₈₉, and VEGF₂₀₆ [16,55,56]. An additional isoform, VEGF-Ax, was also identified in 2014 that arises from programmed translational read-through (PTR) [56]. VEGF₁₆₅a was the first isoform characterised and remains the most extensively investigated in respect to its function, signalling, expression and pathological roles [19]. As a potent stimulator of angiogenesis, VEGF₁₆₅a is considered the prototypical pro-angiogenic VEGF-A isoform. Altered VEGF-A isoform expression has been well documented in tissues during physiological and/or pathological conditions [57,58,59,60,61].

Boundaries between exons are defined by splicing sites which are recognised by a dynamic complex of proteins located in the nucleus called the spliceosome [62], containing five small nuclear ribonucleoproteins (snRNPs)—U1, U2, U4, U5 and U6—plus associated accessory proteins U2AF and SF1. VEGF-A splicing is also regulated by a series of RNA binding proteins, most commonly the serine/arginine (SR) proteins, chiefly SRSF1, SRSF2, SRSF5 and SRSF6 [54]. SR proteins are phosphorylated in the cytoplasm at multiple serine/arginine and proline/serine repeats to enable their subsequent translocation to the nucleus and allows a degree of spatial regulation of splicing. Once in the nucleus, SR proteins typically bind to regulatory sites in VEGF-A pre-mRNA—exonic sequence enhancers [63,64]—which trigger exon removal. One such kinase responsible for phosphorylating SR proteins is the constitutively active kinase SRPK1 [63]. Alterations in SRPK1 expression have been identified (via both upregulation and downregulation) in a range of malignancies. This has led to the development of SRPK1 inhibitors targeting aberrant angiogenesis through altering splicing of endogenous VEGF-A isoforms [63,65,66].

The distinct exons included in each isoform confer different properties (Figure 1 and Figure 2). Exons 1–5 are constitutive exons and are therefore present in all VEGF-A isoforms. These encode a signal sequence (exons 1/2) that is cleaved in the processed form of VEGF, a glycosylation site (Asp74), a potential plasmin cleavage site (Arg110 and Ala111) [67] and residues responsible for VEGFR1 and VEGFR2 binding (Figure 2A,C) [16,68]. A major site of alternative splicing of the Vegfa gene centres on exons 6 and 7. Residues in exons 6a and 7 interact with electronegative heparin sulphate in the extracellular matrix, which has important implications for isoform bioavailability [69,70,71]. The shorter isoforms VEGF₁₁₁ and VEGF₁₂₁ both lack exons 6 and 7, and as a consequence are not tethered to the extracellular matrix (ECM) and are freely diffusible [70,72]. In contrast the longer isoforms VEGF₁₄₅, VEGF₁₈₉ and VEGF₂₀₆ containing both exons 6a and 7 can bind with high affinity to heparin sulphate glycoproteins [73] (Figure 1). The prototypical VEGF₁₆₅a is an intermediate between these freely diffusible and bound isoforms, in that following secretion 50–70% remains cell or ECM bound [72].

A second major site of alternative splicing is driven by the choice of differential 3′ splice acceptor sites within exon 8. In 2002, Bates et al. identified the VEGF_xxxb family of isoforms [74]. These isoforms arise due to distal splicing at a site located 66 base pairs downstream of the proximal splicing site, resulting in isoforms that contain exon 8b (Figure 1 and Figure 2) [51]. In respect to their sequences, VEGF_xxxa and VEGF_xxxb isoforms only differ in the six amino acids found at their C termini; VEGF_xxxa isoforms end in the sequence CDKPRR, whereas VEGF_xxxb isoforms terminate in SLTRKD [75]. Based on both in vitro and in vivo experimental evidence, VEGF_xxxa isoforms are considered to be “pro-angiogenic” as major mediators of vascular permeability, cell proliferation, survival and migration, and angiogenesis [76]; in contrast, VEGF_xxxb isoforms have been reported to have “anti-angiogenic” properties [74,77,78], with evidence that these isoforms may act as regulators and inhibitors of VEGF_xxxa-induced pro-angiogenic activity [51,77]. Interestingly, in quiescent vessels, a higher proportion of total VEGF-A is represented by VEGF₁₆₅b, which is then downregulated in cancer where a switch to pro-angiogenic isoform expression is observed to drive tumour angiogenesis [74,77,79]. Proximal or distal splicing of exon 8 can be influenced by external stimuli, as proximal splicing has been promoted by insulin like growth factor (IGF1) or tumour necrosis factor alpha (TNFα), whereas stimulation with tumour growth factor beta 1 (TGF-β1) has promoted distal splicing [51]. This bias was governed by the specific SR protein splice factor that was bound to a sequence within exon 8a (SRSF1) or 8b (SRSF6). It is worth noting that this bias may not be consistent in all cell types, however this highlights how isoform expression can be context dependent. There has been some debate as to the existence of VEGF_xxxb isoforms physiologically [80,81], with genome wide RNA sequencing data of the human transcriptome questioning whether the relevant exon-exon junctions in the Vegfa gene are present [81].

VEGF-Ax, recently identified by Eswarappa et al. [56], is the result of extended translation beyond the canonical stop codon of VEGF-A mRNA due to the presence of an alternative stop codon within the 3′ untranslated region (Figure 1 and Figure 2). PTR is at least partially regulated by the A2/B1 ribonucleoprotein acting as a trans regulatory factor. The resultant VEGF-Ax therefore contains a 22 amino acid extension encompassing both exons 8a encoded CDKPRR and exon 8b encoded SLTRKD sequences [56,82]. The physiological role of VEGF-Ax is still yet to be fully elucidated with evidence it exhibits both “anti” and “pro-angiogenic” signalling [56,82].

3. VEGF-A Ligand/Receptor Binding

VEGFR2 is a large 151 kDa membrane protein consisting of 7 extracellular immunoglobulin (Ig)-like domains, a single transmembrane helix and a split intracellular kinase domain [83]. VEGF-A is an endogenous agonist for VEGFR2, binding the orthosteric ligand binding site across Ig-like domains 2 (D2) and D3 with a stoichiometry of one VEGF-A dimer across a VEGFR dimer [84,85]. X-ray and NMR structures have identified binding interfaces between VEGF-A and its receptors, confirming key exposed residues at each pole of the homodimer interacting with VEGFR1 [86,87,88] and VEGFR2 [84]. As each VEGF-A isoform contains residues encoded by exons 2–5 (Figure 1), residues interacting with VEGFR1 and VEGFR2 are not removed by alternative splicing (Figure 2A,C). Every isoform also contains cysteine residues that form intermolecular disulphide bonds such that all isoforms are dimeric, as well as forming intramolecular disulphide bonds assembling the Cys-loop folded structure (Figure 2A,C). In contrast, residues identified by structural studies that interact with co-receptor Neuropilin-1 (NRP1) or components of the ECM are absent in some VEGF-A isoforms (Figure 2B,C). The inability to crystallise both the N-terminal (exons 2–5) and C-terminal (exons 6–8) together suggest flexibility between these N- and C-terminal regions of VEGF-A, however the current lack of structural information on full-length VEGF-A isoforms has prevented understanding of the stoichiometry of macromolecular complex assembly.

Ligand binding affinity is the strength of the interaction between a ligand and its receptor, which can be quantified as the concentration of ligand required to bind 50% receptors at equilibrium (equilibrium dissociation constant, K_d) [90]. Traditionally, VEGF-A binding has been investigated in cells expressing VEGFR2 using radiolabelled [¹²⁵I]-VEGF₁₆₅a (

Table 1. Binding affinities of VEGF-A isoforms determined at VEGFR2.

Isoform	Technique	Expression System	Binding Affinity *	Ref.
VEGF165a	Radioligand binding	Human kidney tissue in situ	0.01–0.04 nM	[91]
		HUVECs	0.17 nM	[92]
		Balb/c expressing VEGFR2	0.29 nM	[92]
		COS-1 cells expressing VEGFR2	0.34 nM	[92]
		PAE cells expressing VEGFR2	0.76 nM	[44]
		PAE cells expressing VEGFR2	0.097 nM	[93]
	SPR	VEGFR2 ligand binding domains (D2/D3)	36.7 nM	[85]
	ITC	VEGFR2 ligand binding domains (D2/D3)	18 nM	[85]
		VEGFR2 ligand binding domains (D2/D3)	170 nM	[84]
		VEGFR2 extracellular domain (D1–D7)	2670 nM	[84]
	NanoBRET	HEK293 cells expressing NanoLuc-VEGFR2	0.15 nM	[97]
VEGF165b	NanoBRET	HEK293 cells expressing NanoLuc-VEGFR2	0.39 nM	[97]
VEGF121a	ITC	VEGFR2 extracellular domain (D1–D7)	1120 nM	[84]
		VEGFR2 ligand binding domains (D2/D3)	93 nM	[84]
	NanoBRET	HEK293 cells expressing NanoLuc-VEGFR2	0.34 nM	[97]
VEGF145a	NanoBRET	HEK293 cells expressing NanoLuc-VEGFR2	1.82 nM	[97]
VEGF189a	NanoBRET	HEK293 cells expressing NanoLuc-VEGFR2	1.02 nM	[97]

* Ligand binding affinity quantified as equilibrium dissociation constant of the “hot” ligand (K_d) or competing ligand (K_i). Abbreviations: Bioluminescence resonance energy transfer (BRET) using NanoLuciferase (NanoBRET); isothermal titration calorimetry (ITC); surface plasmon resonance (SPR).

), in which radioligand affinity (K_d) was determined by quantifying bound ligand with saturating concentrations [91,92]. Competing “hot” [¹²⁵I]-VEGF₁₆₅a with increasing concentrations of “cold” ligand allows the determination of affinity of unlabelled VEGF-A isoforms [44,70,77,93] (Table 1). Biochemical techniques have also been used to quantify VEGF-A binding affinities as a cell-free alternative using isolated receptors, including surface plasmon resonance (SPR) [85], solid-phase enzyme-linked assays [56,94] and thermodynamic calorimetry measurements [84,85]. Binding affinities determined using biochemical techniques using truncated VEGFR2 yielded higher estimated binding affinities than those determined with radioligand binding (Table 1), however radioligand binding experiments also have caveats beyond safety and cost. Recently, bioluminescence resonance energy transfer (BRET) was developed as a proximity-based technique using the novel luciferase NanoLuc to monitor ligand binding to GPCRs [95,96]. This allows ligand/receptor interactions to be monitored in real-time to receptors expressed within their native membrane environment. This has been applied to monitor RTK pharmacology, quantifying the binding of single site fluorescently-labelled VEGF₁₆₅a (Figure 3a) to full-length human VEGFR2 in living cells at 37 °C [97].

Regardless of technique used, all VEGF-A isoforms, including those directly comparing “pro-angiogenic” VEGF₁₆₅a and “anti-angiogenic” VEGF₁₆₅b [77,79,97], have been shown to bind to VEGFR2 with nanomolar affinities (Table 1). Relatively lower affinities were seen for VEGF₁₄₅a and VEGF₁₈₉a at VEGFR2 when compared to the prototypical VEGF₁₆₅a, demonstrated by a higher K_d values (Table 1). However, in terms of their pharmacology, these are small differences in affinity (VEGF₁₆₅a K_d 0.15 nM vs. 1.02–1.82 nM; Table 1), particularly as physiological VEGF-A concentrations are estimated within the picomolar range [98]. As all VEGF-A isoforms contain residues that interact with VEGFR2 (Figure 2A,C) and pharmacological binding studies suggest all VEGF-A isoforms bind VEGFR2 with a similar nanomolar affinity (Table 1), this illustrates that VEGF-A/VEGFR2 binding alone is insufficient to explain functional distinctions between isoforms in terms of their signalling downstream of VEGFR2.

4. VEGFR2 Signalling

4.1. VEGFR2 Activation

VEGF-A isoforms have distinct signalling outcomes downstream of VEGFR2 activation [16]. Although VEGF-A isoforms have similar binding properties at VEGFR2, activation of VEGFR2 is a complex multi-step process. As well as VEGF-A binding its orthosteric ligand binding site, allosteric interactions can occur at topographically distinct regions [99]. Allosteric homotypic interactions between VEGFR2 monomers at Ig-like D4, D5 and D7 are an additional step necessary for VEGFR2 activation [84,100,101,102,103], as designed ankyrin repeat protein inhibitors (DARPins) can sterically block these interactions and allosterically inhibit VEGFR2 activation [103,104]. Ligand binding leads to a conformational twist throughout the extracellular region of VEGFR2 reorienting distinct Ig-like domains, shown by electron microscopy [100], small angle X-ray scattering [101], and the full-length crystal structure of structurally related VEGFR1 [88]. VEGF-A binding consequently leads to the rotation of transmembrane helices [105,106,107], with similar configurations induced by isoforms VEGF₁₆₅a, VEGF₁₆₅b and VEGF₁₂₁a when measured using fluorescent resonance energy transfer (FRET) [107]. The intracellular region of VEGFR2 then undergoes conformational changes, formed of N- and C-lobes [108] with ATP binding to the flexible N-lobe cleft which enables receptor intrinsic kinase activity and phosphorylation of tyrosine residues in the C-lobe, notably Y1054 and Y1059 in the activation loop, Y951 in the kinase insert domain and Y1175 and Y1214, respectively [109]. Tyrosine phosphorylation creates binding sites for the recruitment of cytoplasmic adaptor proteins and initiates signalling pathways (reviewed in [37]). Signalling pathways downstream of VEGFR2 activation lead to numerous cellular fates (Figure 4). These include proliferation via PLCγ [110] and ERK1/2 [111], focal adhesion kinase (FAK)-mediated cell migration [112] and cell survival through phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/ protein kinase B (AKT) [113] (Figure 4). VEGFR2 signalling also leads to vascular permeability through FAK recruitment [114,115], p38 MAPK-mediated actin cytoskeleton reorganisation [116] and eNOS activation [117,118]. These signalling pathways undergo extensive cross-talk (reviewed in [42]). VEGFR2 is also dephosphorylated by protein phosphatase 1b (PTP1b) localised to the endoplasmic reticulum [119,120] (Figure 4), highlighting the importance of spatiotemporal trafficking on VEGFR2 activation.

4.2. Distinctions between VEGF-A Isoform Signalling

Functional comparisons of the extent to which VEGF-A isoforms can drive distinct VEGFR2 signalling responses has largely come from phenotypic observations, such as changes in endothelial cell proliferation, or relative levels of phosphorylated VEGFR2/downstream signalling proteins in endothelial cells using Western blots. Despite the importance of VEGF-A concentration on both VEGFR2 binding and signalling (Figure 4), quantifying physiological VEGF-A concentrations is problematic due to the need to consider distinctions between circulating and extracellular VEGF-A, tissue-specific variation, as well as specific VEGF-A isoform concentrations sequestered in the ECM (Section 5.2). Computational modelling of systems pharmacology have predicted a relative expression of VEGF₁₆₅ > VEGF₁₈₉ > VEGF₁₂₁ with total tissue, extracellular or plasma VEGF concentrations below 30 pM [98]. Functional in vitro and in vivo pharmacological experiments have typically quantified responses to agonist stimulation by the relative maximal responses induced, which can give an indication of efficacy, as well as their potency, defined as the concentration of ligand needed to produce an 50% activation/inhibition of the maximal effect (EC₅₀/IC₅₀) inferred from concentration-response curves [90] (Figure 5). Pharmacological investigations of VEGF-A signalling have largely been performed using fixed concentrations of ligand as opposed to full concentration response courses, making it difficult to make direct comparisons of the relative activity of isoforms across different signalling pathways and in different cellular backgrounds. This is due to the nature of agonism requiring knowledge of both affinity and efficacy, as it is not just the affinity of a ligand for its cognate receptor that governs the extent of the signalling response observed. Ligands of equal affinity can produce different maximal responses in functional assays [121,122]. They can also produce different EC₅₀ values if the maximal responses are the same. This is due to the non-linear relationship that exists between receptor occupancy (i.e., the proportion of receptors that are ligand bound) and the final functional responses observed in many cells/tissues as a consequence of signal amplification [121,122]. High efficacy agonists can induce a maximum functional response even at low levels of receptor occupancy (e.g., 20%). Here, a large degree of receptor reserve is seen (i.e., EC₅₀ is much lower the K_d), meaning a high percentage of receptors (e.g., 80%) are not required to produce a maximal response. In contrast, a partial agonist cannot produce a maximal response even when receptor occupancy is 100% where receptor reserve is non-existent (e.g., no “spare” receptors are present). The extent of receptor reserve differs between agonists at the same receptor and between different cell types due to changes in receptor expression level (Figure 5), different agonist efficacies, as well as the coupling efficiency of receptors to signalling partners and the strength of signal amplification. As a result, a ligand that is a partial agonist in a signalling pathway/tissue with low receptor expression (Figure 5B,C) may exhibit full agonism when there is a higher degree of receptor reserve (Figure 5A). A single ligand may therefore show “pluridimensional efficacy” in that it displays a range of efficacies depending on the level of receptor expression, the cell background and signalling pathway observed [123].

As seen from estimations of binding affinity, all VEGF-A isoforms can bind to VEGFR2 with nanomolar affinity (Table 1, Figure 3a). However in vitro and in vivo observations have suggested that stimulation with different VEGF-A isoforms can result in distinct phenotypic outcomes (Figure 3B). Differences between isoforms can therefore not be attributable to affinity alone, but may be explained by differences in their relative intrinsic efficacies and the impact of receptor expression and signalling efficiency in different cells and tissues. The starkest of these phenotypic differences is the classification of VEGF-A isoforms into “pro-angiogenic” VEGF_xxxa or “anti-angiogenic” VEGF_xxxb groups. VEGF₁₆₅a is the prototypical VEGF-A isoform that has been shown to act as a full agonist for VEGFR2 driven signalling responses observed both in vivo and in vitro and is therefore typically used as a reference ligand for investigating other VEGF-A isoforms [70,77,78,79,124,125,126,127,128,129,130,131,132]. Of the selective isoforms studied, VEGF₁₆₅a has been shown to induce the highest levels of phosphorylation of VEGFR2 (Y1175 residue), AKT and ERK [132].

Since its discovery in 2002 [74], the VEGF₁₆₅b isoform has been characterised as “anti-angiogenic” in relation to the prototypical VEGF₁₆₅a. VEGF₁₆₅b stimulation results in a reduced stimulation of angiogenesis in vivo when compared to VEGF₁₆₅a [77,79], as well as lower tumour vessel density [133], endothelial cell proliferation [78,125,126,134], tubulogenesis and wound healing in vitro [134]. These data are consistent with VEGF₁₆₅b acting as a low efficacy agonist in some signalling pathways rather than acting as an antagonist lacking any ability to activate VEGFR2 signalling. However, for responses in cells where VEGF₁₆₅b is clearly acting as a weak partial agonist it will be able to antagonise the agonist effect of more efficacious agonists (e.g., VEGF₁₆₅a) (Figure 5D). This is supported by biochemical observations that VEGF₁₆₅b can induce phosphorylation of VEGFR2, albeit to a decreased level when compared to the full agonist VEGF₁₆₅a [125,128], suggesting that VEGF₁₆₅b can still partially activate VEGFR2. Additionally, VEGF₁₆₅b can stimulate the production of NFAT in HEK293 cells expressing VEGFR2 alone, with a comparable potency to VEGF₁₆₅a (EC₅₀ VEGF₁₆₅a 0.1 nM vs. VEGF₁₆₅b 0.4 nM; Figure 3b) [97] albeit with decreased efficacy in respect to VEGF₁₆₅a (68.1% ± 5.7 of maximal VEGF₁₆₅a response; Figure 3B) [97]. Co-stimulation of VEGF₁₆₅a with VEGF₁₆₅b also leads to decreased maximal responses compared to VEGF₁₆₅a alone both in vivo and in endothelial cells indicative of VEGF₁₆₅b partial agonism [77,79]. Phenotypically, decreased vascular permeability has also been observed with VEGF₁₆₅b treatment compared to VEGF₁₆₅a measured by both transendothelial electrical resistance and following macromolecules conjugated to fluorescent FITC [128,129]. Cell-specific differences have been seen in respect to VEGF₁₆₅b induced phosphorylation of AKT in HMVECs [77] and HCAECs [134] where it is decreased when compared to VEGF₁₆₅a, but is comparable to VEGF₁₆₅a in HPMECs [128]. Additionally, whilst differences in the extent of ERK phosphorylation were seen with VEGF₁₆₅b vs. VEGF₁₆₅a in HPMECs [128] and PAECs (expressing VEGFR2) [126], the levels of pERK were indistinguishable in HMVECs [77]. These differences in efficacy illustrate how the nature of VEGF-A agonism may be heavily influenced by the expression level or localisation of VEGFR2, but also other VEGF-A binding partners (VEGFR1, NRP1) or downstream adaptor proteins both in different physiological and pathological cell systems [133].

For the shorter freely diffusible VEGF-A isoform, VEGF₁₂₁a (Figure 1), evidence exists of it exhibiting both partial and full agonism (in comparison to VEGF₁₆₅a) depending on the signalling pathway observed. In both in vivo angiogenesis [79,125] and in vitro measurements of signalling (Figure 3b), VEGF₁₂₁a appears to act as a partial agonist in comparison to VEGF₁₆₅a. VEGF₁₂₁a stimulation also leads to submaximal HUVEC proliferation when compared to VEGF₁₆₅a [124,126,130,132], with both a rightward shift in potency and reduced maximal response also seen [67]. Compared to VEGF₁₆₅a, VEGF₁₂₁a also induced less HUVEC motility and sprouting [124], as well as partial calcium signalling responses [97,131]. Western blots performed by several independent groups have also suggested VEGF₁₂₁a stimulation induces reduced phosphorylation of VEGFR2 directly [20,124,130], as well as ERK [126] and PLCγ [131] when compared to VEGF₁₆₅a. In respect to NFAT production, VEGF₁₂₁a showed comparable potency to VEGF₁₆₅a (0.3 vs. 0.1 nM respectively; Figure 3B) but reduced efficacy (65.9% ± 8.8 of maximal VEGF₁₆₅a response; Figure 3b) [97]. However, evidence exists that the extent of VEGF₁₂₁a agonism is pathway dependent, for example, VEGF₁₂₁a-induced ex vivo angiogenic sprouting has been shown as both comparable [70] and lower [20] than VEGF₁₆₅a, with similar trends also seen in respect to vascular permeability seen as both comparable [132,135] and lower [136] than VEGF₁₆₅a.

The ECM-bound isoforms, VEGF₁₄₅a and VEGF₁₈₉a, also show variations in the extent of agonism at VEGFR2 depending on the signalling pathway observed. Relative to VEGF₁₆₅a, VEGF₁₄₅a is less able to stimulate angiogenesis in vivo, albeit to a greater extent than observed with VEGF₁₂₁a or VEGF₁₆₅b stimulation [125]. VEGF₁₄₅a-induced HUVEC proliferation and permeability was also partial relative to VEGF₁₆₅a [132], however this same study also found VEGF₁₄₅a-induced HUVEC migration was comparable to VEGF₁₆₅a [132]. Reduced VEGFR2 phosphorylation has been observed in both murine endothelial cells [125] and HUVECs [132], as well as reduced ERK or AKT phosphorylation [132]. Although VEGF₁₈₉a had similar functional activity to VEGF₁₆₅a in terms of concentration-dependent proliferation in HUVECs and migration of BAECs [127], autocrine expression in isolation revealed distinctions in proliferation and cell survival [137]. Furthermore, in HEK293 cells lacking NRP1 expression and solely expressing VEGFR2, both VEGF₁₄₅a and VEGF₁₈₉a showed comparable potencies to VEGF₁₆₅a in respect to NFAT production (Figure 3b) albeit with decreased efficacy (71–72% of maximal VEGF₁₆₅a response; Figure 3b) [97].

Following its identification in 2014 [56], the novel isoform VEGF-Ax has also been characterised as “anti-angiogenic” due to its functional similarities to VEGF₁₆₅b. Based on the early in vitro and in vivo evidence generated so far, VEGF-Ax has shown evidence of both full and partial agonism depending on the signalling pathway investigated. VEGF-Ax induced a decreased maximum response relative to VEGF₁₆₅a in respect to VEGFR2 phosphorylation in HUVECs [56]. However, VEGF-Ax has also been shown to induce vascular permeability and ex vivo migration of HUVECs as well as promote BAEC proliferation to a comparable extent seen with VEGF₁₆₅a stimulation [82]. VEGF-Ax possesses functional similarities to the VEGF_xxxb isoforms, although questions remain over how frequently posttranslational read-through occurs to bypass the canonical stop codon and if this process occurs in all cells equally.

5. Molecular Mechanisms Distinguishing between VEGF-A Isoforms

VEGFR2 is subject to complex regulation by numerous mechanisms that underlie context-dependent signalling. It is therefore unsurprising that endogenous isoforms of varying lengths or sequences have distinct signalling outcomes with implications for altered expression in health and disease [16]. The spatial and temporal regulation of both VEGFR2 in terms of trafficking, and VEGF-A isoform bioavailability through ECM interactions, can influence isoform-specific signalling. Endothelial cells also express interaction partners NRP1 [138] and integrins [139], as well as other modulatory membrane and cytoplasmic proteins that may alter receptor expression or localisation, ultimately influencing downstream signalling and phenotypic outcomes that distinguish between VEGF-A isoforms.

5.1. Spatiotemporal Dynamics of VEGFR2 Trafficking

As demonstrated for other RTKs and G protein coupled receptors (reviewed in [140]), VEGFR2 undergoes endosomal signalling as it can signal from both the plasma membrane and intracellular compartments [42,132,141]. Activation of ERK requires VEGFR2 endocytosis [141], however the off-target effects of endocytic inhibitors on ERK activation requires careful interpretation [142]. VEGFR2 is localised at both the plasma membrane and in early endosomes due to constitutive VEGFR2 internalisation and recycling [143,144]. VEGFR2 can be internalised via clathrin-dependent [145,146] and -independent mechanisms [147,148]. Following VEGF-A stimulation, ligand-receptor complexes undergo internalisation within 15–20 min [97,149]. Ligand stimulation also triggers VEGFR2 recycling back to the plasma membrane [150], via short loop Rab4-positive endosomes or long loop Rab11-positive endosomes [144] (Figure 4). Alternatively, ubiquitination can initiate proteolysis and trafficking of VEGFR2 for lysosomal degradation [149,151] (Figure 4). The intracellular fate of VEGFR2—recycling or degradation—regulates the duration, amplitude and specificity of the signalling response [38,152], as the presence of activated VEGFR2 in early endosomes induces maximal ERK1/2 and AKT activation while p38 MAPK signal transduction is dependent on cell surface VEGFR2 expression [38]. Subcellular VEGFR2 trafficking is also important for receptor dephosphorylation due to the intracellular localisation of the key regulator protein phosphatase 1b (PTP1b) [119,120]. Crucially, some evidence has suggested isoform-specific trafficking of VEGFR2 [132,152], whereby VEGF₁₆₅a induces a greater degree of ubiquitinylation than the partial agonists VEGF₁₂₁a or VEGF₁₄₅a [132]. Additionally, VEGFR2 internalisation can be modulated by NRP1, a co-receptor that only selected VEGF-A isoforms can interact with, rerouting VEGFR2 to long-loop recycling endosomes rather than degradative pathways [153]. VEGFR2 trafficking can also be modulated by membrane proteins VE-cadherin and ephrin B2 [145,154], however it is unknown whether these preferentially interact with specific VEGF-A isoforms. Factors modulating the spatiotemporal dynamics of VEGFR2 trafficking combined with evidence of endosomal signalling suggest a possible mechanism which may drive VEGF-A isoform specific altered signalling.

5.2. Spatiotemporal Dynamics of VEGF-A Bioavailability

Regulation of the bioavailability of VEGF-A isoforms following secretion may contribute to their differential biological and cellular responses [16,42]. VEGF-A isoform bioavailability is heavily influenced by their differing abilities to interact with the ECM (Figure 1). Tethering to the ECM creates localised concentrations of VEGF-A close to cells, which can be proteolytically released or cleaved to generate shorter more diffusible isoforms, creating VEGF-A gradients that can amplify VEGF-A signalling in times of angiogenic need [155]. Residues encoded by exon 6a, present in VEGF₁₄₅, VEGF₁₈₉ and VEGF₂₀₆ (Figure 1), as well as exon 7-encoded residues, can interact with electronegative heparin through arginine residues confirmed by mutagenesis [70] and an NMR solution structure [156] (Figure 2B,C). Despite lacking exon 6a-encoded residues, VEGF₁₆₅a binds heparin with relatively high affinity through exon 7-encoded arginine residues (K_d = 40–157 nM) [157] (Figure 2B,C). In addition to its role in sequestering VEGF-A isoforms, heparin can potentiate binding of VEGF₁₆₅a to VEGFR2, but not freely diffusible VEGF₁₂₁a as it lacks exon 6/7 [158,159,160]. Using SPR, Teran and Nugent (2015) did not observe any binding of VEGFR2 itself to heparin, leading them to postulate that VEGF₁₆₅a bridges VEGFR2 and heparin so that they form a larger complex [160]. Functionally the presence of heparin increases VEGF₁₆₅a, but not VEGF₁₂₁a, mediated phosphorylation of VEGFR2 and enhanced VEGF₁₆₅a-induced HUVEC mitogenesis in a dose-dependent manner [161]. Heparin also was shown to substantially enhance the affinity of interactions between VEGF₁₆₅a and co-receptor NRP1 [162,163], illustrating the interplay between these modulatory factors on VEGF-A bioavailability and pharmacology.

5.3. Interactions with Co-Receptor Neuropilin-1 (NRP1)

VEGFR2 signalling is selectively enhanced by its co-receptor NRP1 [164,165], a multifaceted single transmembrane glycoprotein with which only some VEGF-A isoforms can bind (Figure 2b,c). NRP1 is also involved in neuronal guidance through binding structurally and functionally unrelated class 3 semaphorins at a distinct extracellular domain [163,166], however its functional role in vessel development is evident from the severe cardiovascular abnormalities exhibited in Nrp1 knockout mice [167,168,169]. As with VEGFR2 [170], NRP1 upregulation in malignant tumours is correlated to aggressive cancer phenotypes [170,171,172]. NRP1 selectively potentiates VEGFR2-mediated endothelial cell motility and vascular permeability with minimal effect on proliferation, driving arterial vessel development in vivo [173,174,175]. Molecular mechanisms enhancing VEGFR2 signalling were further elucidated following the use of antibodies [124] or siRNA [20] that blocked NRP1 leading to reductions in VEGF₁₆₅a-induced VEGFR2 and ERK phosphorylation, respectively, in vitro. VEGF-A binds the b1 domain of NRP1 [89,162,176] at exposed Tyr297 and Asp320 residue sidechains [89,177,178], primarily via an exon 8a-encoded arginine residue (CDKPRR; critical arginine underlined) with homologous interacting residues in tuftsin (TKPR) [176,179] and peptide ATWLPRR [180,181]. VEGF₁₆₅a has a comparable binding affinity for NRP1 compared to VEGFR2 determined by radioligand binding (K_d < 3 nM) [92,159], while cell-free SPR-derived K_d values were ~100-fold higher [124,182]. With no direct VEGFR2/NRP1 binding interface identified, NRP1 is thought to modulate VEGFR2 through forming a multimeric complex bridged by NRP-binding VEGF-A isoforms. These dimeric VEGF-A ligands then interact with VEGFR2 and NRP1 via separate ends of the peptide [165,183] (Figure 2). HUVECs express more NRP1 than VEGFR2, quantified as 35,000 NRP1 receptors compared to 6000 VEGFR2 receptors per cell [152]. While NRP1-mediated VEGFR2 modulation is typically thought to arise from receptors expressed in the same cell, NRP1 expressed by adjacent cells can have distinct effects on VEGFR2 signalling, adding another layer of complexity [184]. NRP1 has a 44-amino acid intracellular domain which lacks catalytic activity, suggesting it may not be capable of signalling in its own right. However, its short cytoplasmic tail does contain a Serine-Glutamate-Alanine motif that interacts with PDZ domain-containing synectin [185,186,187]. NRP1 is suggested to modulate VEGFR2 trafficking or expression [153], likely via synectin-mediated myosin VI recruitment which is capable of transporting internalised receptors along actin filaments towards the cell body [188,189,190,191]. Interestingly VEGF-A isoforms show differences in respect to NRP binding. VEGF₁₆₅b [79,126] and VEGF-Ax [56,82] cannot bind NRP1, whereas VEGF₁₆₅a [79,89,92,124,125,126,159,182,192] and VEGF₁₈₉a [193] containing exons 7- and 8a-encoded residues can bind NRP1. There are conflicting reports regarding whether VEGF₁₂₁a, the shorter VEGF-A isoform containing exon 8a-encoded CDKPRR but lacking exon 7, can bind NRP1 (reviewed in [194]). While cell-free assays measured low affinity binding [89,124,126], other assays have not detected any interaction between VEGF₁₂₁a and NRP1 [79,82,125], suggesting VEGF₁₂₁a may be unable to bridge the multimeric NRP1/VEGFR2 complex. Interestingly despite containing both exon 8a-encoded (CDKPRR) and the exon 8b-encoded (SLTRKD), VEGF-Ax is unable to bind NRP1 [56,82], with suggestions that this may be due to the 22-amino acid extension present in VEGF-Ax sterically blocking binding [82]. The ability for heparin to synergistically enhance VEGF/VEGFR2/NRP1 complex formation [160] in conjunction with the differential abilities of VEGF-A isoforms to interact with NRP1, adds another layer of complexity to the VEGF/VEGFR2 signalling axis.

5.4. Heterodimer Formation with VEGFR1

Another potential mechanism that further diversifies VEGF/VEGFR2 signalling outcomes, is via the formation of heteroreceptor complexes between VEGFR2 and other VEGFR subtypes [195,196,197,198,199]. VEGFR2 mediated endothelial cell proliferation is negatively regulated by membrane-bound VEGFR1 homodimers [195,200]. There is a substantial decrease in VEGFR1 vs. VEGFR2 expression on the surface of endothelial cells [138], therefore VEGFR1 homodimers are likely to be relatively rare when compared to VEGFR1/VEGFR2 heterodimers [196]. Computational simulations suggest 10–50% VEGFR2 monomers are likely to exist as preassembled VEGFR1/VEGFR2 heterodimers during basal conditions with no increase upon ligand stimulation [196,201]. Enzyme-linked immunosorbent assays (ELISA) have identified the presence of preassembled VEGFR1/VEGFR2 complexes in highly vascularised organs in mice, including lung, kidney and liver [196]. These preassembled complexes have also been documented in primary cell lines, including bovine [202], porcine [203] and murine [204] endothelial cells. The use of a novel dimeric bivalent ligand (VEGF-E/PIGF-1) formed by a VEGF-E monomer (specific-ligand for VEGFR2) and a PIGF-1 monomer (specific-ligand for VEGFR1), allowed the selective activation of VEGFR1/VEGFR2 heterodimers [196]. Stimulation with VEGF-E/PIGF-1 ligand in HUVECs led to VEGFR2 phosphorylation, but relatively weak ERK1/2 phosphorylation and intracellular calcium mobilisation, compared to VEGF-A and VEGF-E alone. Additionally, VEGF-E/PIGF-1 stimulation promoted endothelial cell migration, sustained in vitro tube formation and vasodilation, but failed to mediate proliferation and endothelial factor production, suggesting that mediation of these processes may be bias towards VEGFR2 homodimers [196]. Moreover, these VEGFR1/VEGFR2 complexes also inhibited VEGF-A-induced prostacyclin release, and phosphorylation of VEGFR2 was greater in cells lacking VEGFR1, suggesting VEGFR1 may negatively modulate VEGFR2 activity in endothelial cells [196]. VEGFR2/VEGFR3 heterodimers have also been identified using proximity ligation assays [197,198,199], however, compared to VEGF-C, VEGF-A did not enhance VEGFR2/VEGFR3 heterodimer association [197]. The phosphorylation pattern for VEGFR3 homodimers and VEGFR2/3 heterodimers also showed differences [197,198,199], suggesting specific tyrosine residues may be exclusive substrates for VEGFR3 homodimers [197] and that VEGFR2/VEGFR3 heterodimers may show distinct signalling transduction and biological properties. Interestingly VEGF-A isoforms with higher affinity for heparin, such as VEGF₁₄₅ and VEGF₁₈₉, further potentiated VEGFR2/VEGFR3 heterodimer association when compared to VEGF₁₆₅a or VEGF₁₂₁a [198], providing evidence of isoform-specific heterodimer formation with functional consequences on signalling outcomes.

6. Conclusions and Future Perspectives

VEGF-A isoforms are distinct endogenous agonists for VEGFR2 that give rise to different functional outcomes despite similar binding properties at VEGFR2. Considering prototypical VEGF₁₆₅a as a full agonist which stimulates maximal responses, several groups have provided evidence that other VEGF-A isoforms are partial agonists in stimulating a sub-maximal response relative to VEGF₁₆₅a, including VEGF₁₆₅b, VEGF₁₂₁a, VEGF₁₄₅a and VEGF-Ax. In terms of their molecular pharmacology, the potency and efficacy of signalling responses are both pathway- and context-dependent and heavily influenced by receptor expression and signalling protein coupling efficiency. Mechanisms distinguishing between VEGF-A isoforms, including ECM and NRP1 interactions, highlight the importance of considering VEGF-A/VEGFR2 signalling with spatiotemporal resolution. Applying quantitative pharmacological techniques used extensively with other cell surface receptor families, such as G protein coupled receptors, could further inform our molecular understanding of the VEGF/VEGFR signalling axis. The use of new technologies such as CRISPR/Cas9 would allow quantitative pharmacological observations to be performed in different cellular contexts at physiological expression levels. Endogenous VEGF-A isoform expression has been shown to be dependent on the tissue, disease state and splicing factors present, however fundamental questions remain concerning how these isoforms lead to nuances of signalling at a molecular level.

VEGF-A is critical in the development of a number of angiogenesis-dependent conditions, such as endometriosis [205], diabetic nephropathy [206], retinopathy [10], neuropathy [207], pulmonary fibrosis [61], neovascular eye diseases [208] and ischaemic heart disorders [209,210,211], as well as numerous cancer types as angiogenesis is a common hallmark for tumour development [6,212,213]. VEGF-A/VEGFR2-mediated signalling is targeted through neutralising circulating VEGF-A using bevacizumab (Avastin), blocking its receptors with RTK inhibitors (RTKIs) or inhibiting downstream signalling pathways [214,215,216]. Existing approaches lack isoform specificity, as the epitope of bevacizumab binds the VEGFR-binding region of VEGF-A (Figure 1 and Figure 2) that are present in all isoforms [217,218]. Recently, SRPK1 inhibitors have been developed to modulate VEGF-A isoform expression through favouring splicing towards partial agonist VEGF₁₆₅b rather than full agonist VEGF₁₆₅a [63,65]. Crucially, approved therapeutics used in oncology largely lack long-term efficacy due to the recurrent emergence of resistance mechanisms [219,220,221]. Anti-cancer therapeutics targeting VEGF-A/VEGFR2 are often used in combination with chemotherapy [25]; as a genotoxic therapeutic agent, chemotherapeutics may promote splicing to VEGF₁₁₁ [50]. Numerous RTKIs also have on-target adverse effects due to the scope of VEGFR signalling pathways, such as hypertension caused by inhibiting pathways leading to vascular permeability [222] and vasoconstriction [223]. Molecular pharmacology forms the basis of drug development, therefore further elucidating the mechanisms that distinguish between VEGF-A isoform pharmacology and how these are orchestrated in health and in disease is fundamental to developing novel ways of targeting VEGF/VEGF receptors.