The VEGF-A locus: a gene with two faces
Vascular endothelial growth factor A (VEGF-A) is the founding member of the VEGF family and the dominant regulator of physiological and pathological angiogenesis — the growth of new blood vessels from existing ones. Its gene spans eight exons and generates multiple isoforms through alternative splicing of exons 6, 7, and 8. The well-known isoforms — VEGF121, VEGF165, VEGF189, VEGF206 — differ in how much of exons 6 and 7 they include, which determines their heparin-binding affinity and consequently their diffusibility in extracellular matrix. A VEGF165 molecule that binds strongly to heparan sulfate proteoglycans in the extracellular matrix forms a local gradient; VEGF121, lacking heparin-binding domains, diffuses more freely.
The crucial and less widely appreciated dimension of VEGF-A splicing concerns exon 8 — the terminal coding exon. Exon 8 has two functional splice acceptor sites: a proximal site, 66 nucleotides upstream from the distal site. Usage of the proximal splice site produces the standard pro-angiogenic isoforms (VEGF165, VEGF121, etc.) with the conventional C-terminal peptide encoded by the proximal exon 8 sequence. Usage of the distal splice site produces the VEGFxxxb family — VEGF165b, VEGF121b, VEGF189b — which share an identical N-terminal sequence (including the VEGFR2 binding domain) with their standard counterparts but have a different, six-amino-acid C-terminal peptide encoded by the distal exon 8 reading frame.
This six-amino-acid difference in the C-terminus — the VEGFxxxb family ends with …SLTRKD rather than …CDKPRR — has profound functional consequences. The canonical C-terminal sequence of standard VEGF isoforms is critical for the neuropilin-1 (NRP1) co-receptor interaction and for a specific receptor kinase activation conformation at VEGFR2. VEGFxxxb isoforms retain full binding affinity to VEGFR2 — they compete with VEGF165 for the receptor — but do not activate VEGFR2 to the same extent. They function as partial agonists or effective antagonists of VEGFR2 signalling, reducing downstream activation of AKT, ERK, and the cascade of events promoting endothelial cell proliferation and migration.
The VEGFxxxb family: VEGF121b, VEGF165b, VEGF189b
VEGF165b is the best-characterised member of the anti-angiogenic VEGFxxxb family and for many years was the only recognised member. Work from the laboratory of David Bates and colleagues at the University of Bristol over the 2000s and 2010s progressively characterised VEGF165b, demonstrating its expression in normal tissues (particularly kidney glomeruli, retinal pigment epithelium, and anterior chamber of the eye), its reduction in pathological angiogenic states, and its anti-angiogenic activity in experimental models. Later work confirmed VEGF121b and VEGF189b as similarly functional anti-angiogenic isoforms.
In normal adult vasculature, VEGFxxxb isoforms contribute substantially to total VEGF-A protein — in some normal tissues the VEGFxxxb/total VEGF-A ratio is 50% or higher, providing a constitutive brake on unnecessary angiogenesis. This ratio shifts dramatically in pathological conditions. In tumours, particularly colon cancer, renal cell carcinoma, and breast cancer, the VEGFxxxb fraction drops to as low as 10–20% of total VEGF — the shift toward the pro-angiogenic isoforms supports tumour neovascularisation. In age-related macular degeneration (AMD) and diabetic retinopathy, VEGFxxxb is reduced in the vitreous and retina, permitting uncontrolled choroidal neovascularisation and retinal neovascularisation respectively.
The clinical implication is significant. Current anti-VEGF therapy for AMD and for cancer — using antibodies or fragments that block all VEGF-A isoforms (ranibizumab, bevacizumab, aflibercept) — blocks both pro-angiogenic and the residual anti-angiogenic VEGFxxxb. Some researchers have proposed that selectively restoring VEGFxxxb levels, rather than globally blocking VEGF-A, might be a more targeted approach — maintaining the beneficial vascular maintenance signals of VEGF while exploiting its own endogenous antagonists. This remains a therapeutic hypothesis in preclinical development.
SRSF1, SRSF2, and the splicing code for exon 8
The choice between the proximal (pro-angiogenic) and distal (anti-angiogenic) splice sites of VEGF exon 8 is controlled by serine/arginine-rich (SR) splicing factors. SR proteins bind to exonic splicing enhancer sequences and facilitate spliceosome assembly at adjacent splice sites. Two SR proteins are particularly important for the VEGF exon 8 splicing decision.
SRSF1 (also called SF2/ASF), when expressed at high levels, promotes usage of the proximal splice site, producing the standard pro-angiogenic isoforms. SRSF1 is overexpressed in many cancers and is considered a proto-oncogenic splicing factor — its effects on VEGF splicing are one of several pro-tumorigenic splicing changes it promotes. The mechanism involves SRSF1 binding to an exonic splicing enhancer in the proximal region of exon 8 and facilitating U1 snRNP and U2AF binding to the proximal splice site.
SRSF2 and its upstream regulator CLK1 (CDC-like kinase 1) tend to promote the distal splice site and anti-angiogenic isoform production. CLK1 phosphorylates SRSF2, modulating its activity and its relative balance with SRSF1. CLK1 inhibition or SRSF2 depletion shifts splicing toward the pro-angiogenic proximal splice site; conversely, conditions that activate CLK1 or favour SRSF2 can shift the balance toward VEGFxxxb production.
The SR protein splicing network also involves SRPK1 and SRPK2 (SR protein kinases), which phosphorylate SR proteins in the cytoplasm prior to their nuclear import. Hyperphosphorylated SR proteins are transported to the nucleus but must be partially dephosphorylated by the nuclear phosphatase PP1 to become active splicing factors. The balance of SR protein phosphorylation state — influenced by SRPK activity, PP1 activity, and CLK1 activity — determines which splice sites are preferred in a given cellular context. This phosphorylation balance is sensitive to cellular redox state and inflammatory signalling, making the VEGFxxxb splicing decision potentially responsive to antioxidant interventions.
VEGF splicing in AMD and diabetic retinopathy
In the healthy retina, VEGF165b is the predominant VEGF isoform in retinal ganglion cells and retinal pigment epithelium (RPE). It likely serves a neuroprotective role — VEGFR2 activation in neurons can promote neuronal survival — while simultaneously keeping the underlying choroidal vasculature in a quiescent state. This balance is disrupted in AMD. Immunohistochemical analysis of AMD retinas shows reduced VEGF165b in RPE and outer nuclear layer, coinciding with the choroidal neovascularisation that characterises wet AMD.
In diabetic retinopathy, VEGF165b was found to be reduced in vitreous samples from patients with active proliferative retinopathy compared to controls, and in animal models of oxygen-induced retinopathy (OIR), VEGF165b treatment reduced pathological neovascularisation without affecting physiological vascular development. Intravitreal injection of VEGF165b protein has been studied as a potential therapeutic modality in these retinal conditions — conceptually distinct from anti-VEGF blockade because it restores the endogenous anti-angiogenic signal rather than suppressing all VEGF signalling.
VEGF splicing in tumour angiogenesis
Tumour angiogenesis requires sustained VEGF signalling to support the developing vascular network that feeds tumour growth beyond approximately 1–2 mm in diameter — the diffusion limit for oxygen and nutrients. Tumours achieve high VEGF output through a combination of transcriptional upregulation (via HIF-1α under hypoxia) and splicing shift toward pro-angiogenic isoforms. The SRSF1 overexpression frequently seen in solid tumours is one mechanism for the splicing shift; others include loss of expression of splicing factors that support the distal splice site.
The observation that VEGFxxxb isoforms are reduced in many tumours — and that restoring their expression or exogenously administering VEGF165b protein reduces tumour angiogenesis and growth in experimental models — has attracted interest as an anti-cancer strategy. The challenge is that the same VEGF-A molecule provides pro-survival signals to endothelial cells and possibly to tumour cells directly, making isoform-selective interventions more attractive in principle than total VEGF blockade.
Oxidative modification of SR proteins: the redox-splicing link
The molecular link between cellular redox state and alternative splicing is an active area of research. Several mechanisms have been identified. SR proteins, including SRSF1 and SRSF2, contain cysteine residues that can be oxidatively modified — S-glutathionylation, S-nitrosylation, and sulfenic acid formation at these cysteines can alter protein conformation, RNA binding specificity, and nuclear-cytoplasmic shuttling. Under conditions of elevated reactive oxygen species (ROS), certain SR proteins are redistributed from nuclear speckles (where active splicing occurs) to the cytoplasm, or undergo conformational changes that alter their splice site preferences.
In the context of VEGF exon 8 splicing, this means that elevated cellular ROS — the characteristic environment of many tumour cells and of the ageing retina — could contribute to the shift toward pro-angiogenic isoforms by modifying SRSF1/SRSF2 activity and the SRPK/CLK1 phosphorylation balance. Conversely, reducing oxidative stress pharmacologically or nutritionally could, in principle, shift the SR protein environment toward one that favours VEGFxxxb production.
Phycocyanin — the primary antioxidant constituent of spirulina — is a potent scavenger of peroxyl radicals, hydroxyl radicals, and peroxynitrite, and acts as an inhibitor of NADPH oxidase (Nox2). In cell culture models, phycocyanin and phycocyanobilin reduce superoxide production and protect cells from oxidative damage across concentrations that are physiologically achievable with supplementation. The logical chain is: spirulina supplementation → phycocyanin absorption → reduced ROS in tissues → less oxidative modification of SR proteins → altered SRSF1/SRSF2 balance → potentially increased VEGFxxxb/VEGF165 ratio.
This chain of reasoning is plausible but speculative. Each step has supporting evidence individually, but the full chain — from spirulina ingestion to VEGF isoform ratio change in tumour or retinal tissue — has not been demonstrated experimentally. The effect size, if it exists, is unknown. The mechanism requires that phycocyanin metabolites reach the relevant tissue (retina, tumour) in sufficient concentration to meaningfully alter ROS levels in the nucleus where splicing occurs — a non-trivial requirement.
Spirulina and anti-angiogenic activity: the existing evidence
Setting aside the VEGF splicing hypothesis, spirulina has documented anti-angiogenic properties in experimental systems independent of any specific mechanism. Phycocyanin has been shown to reduce tumour vascularity in xenograft mouse models, and several in vitro studies have demonstrated that spirulina extracts reduce endothelial cell tube formation (a surrogate for angiogenesis in culture) and reduce VEGF production by tumour cells exposed to phycocyanin.
The mechanisms invoked in these studies include: NF-κB inhibition reducing VEGF transcription (HIF-1α and NF-κB both drive VEGF promoter activity); direct scavenging of the ROS that stabilise HIF-1α under normoxia (the pseudohypoxic state common in cancer); and modulation of the PI3K/AKT/mTOR pathway that controls HIF-1α translation. The VEGFxxxb splicing route is a speculative additional mechanism that could act in parallel with these more established pathways.
For retinal diseases specifically — AMD and diabetic retinopathy — human clinical trial data on spirulina are essentially absent. The plausibility of a VEGFxxxb-restoring effect via antioxidant action is interesting enough to warrant investigation in retinal cell models (ARPE-19 cells expressing VEGF isoforms under oxidative stress conditions) as a first step, but there is currently no clinical basis for recommending spirulina supplementation as an adjunct to AMD therapy.
What the evidence supports and what it does not
The evidence strongly supports: the existence of VEGFxxxb anti-angiogenic isoforms; their reduction in AMD, diabetic retinopathy, and several cancer types; the control of their production by SRSF1/SRSF2 splicing factors; and the sensitivity of SR protein activity to cellular redox state. The evidence also supports phycocyanin’s antioxidant activity and anti-angiogenic effects in experimental models.
What the evidence does not yet support is the specific causal connection: that spirulina supplementation shifts VEGF isoform splicing toward VEGFxxxb in human tissues, in vivo, at relevant doses. This is a testable hypothesis — VEGF isoform profiling in plasma or tissue biopsies after spirulina supplementation in a clinical cohort would be a straightforward experiment — but it has not been performed. The gap between mechanistic plausibility and demonstrated clinical effect is an honest characterisation of where this research stands.
For readers interested in spirulina and eye health or cancer biology, the most accurate summary is: spirulina has real anti-angiogenic properties in experimental systems, the mechanism likely involves multiple pathways simultaneously including HIF-1α/NF-κB suppression and possibly VEGF isoform splicing, but the VEGFxxxb splicing angle is speculative and not yet demonstrated in vivo.