The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.

You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.

We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.

We are interested in where in or outside the cell the gene product carries out its function.

We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.

Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.

Research Report: Human VEGFA (UniProt P15692) Functional Annotation and Translation

Target verification (mandatory): UniProt accession P15692 corresponds to human VEGFA (vascular endothelial growth factor A; VEGF-A), a member of the PDGF/VEGF growth factor family and the prototypic VEGF ligand driving angiogenesis through VEGF receptors. VEGFA biology includes (i) secreted VEGF-A isoforms produced by alternative splicing, and (ii) a hypoxia-associated non-canonical long form (L-VEGF) that can be proteolytically processed into a secreted VEGF-A fragment plus a distinct nuclear N-terminal fragment (N-VEGF). (perezgutierrez2023biologyandtherapeutic pages 1-3, katsman2022nvegftheautoregulatory pages 1-3)

1) Key concepts, definitions, and current understanding

1.1 Core definition and primary function

VEGF-A is a secreted dimeric growth factor whose primary physiological role is to activate endothelial cells to support vasculogenesis/angiogenesis (development and remodeling of blood vessels) and to regulate vascular permeability. Mechanistically, VEGF-A ligands signal mainly through receptor tyrosine kinases VEGFR1 (FLT1) and VEGFR2 (KDR/FLK1), with VEGFR2 functioning as the main pro-angiogenic signaling receptor. (perezgutierrez2023biologyandtherapeutic pages 1-3, perezgutierrez2023biologyandtherapeutic pages 5-6)

A key concept in VEGFA functional annotation is that it is not a single molecular species: alternative splicing, extracellular processing, and even non-canonical translation generate forms with different diffusion/ECM retention properties and potentially different biological outputs. (perezgutierrez2023biologyandtherapeutic pages 1-3, perezgutierrez2023biologyandtherapeutic pages 17-17)

1.2 Isoforms and processed products (secreted VEGF-A and L-VEGF/N-VEGF axis)

Alternative splicing (canonical secreted isoforms): VEGFA is encoded by a single 8-exon gene that yields multiple secreted isoforms, classically including VEGF121, VEGF165, VEGF189, VEGF206. Isoform-specific inclusion of heparan-binding features drives different degrees of extracellular matrix (ECM)/heparan sulfate proteoglycan (HSPG) binding versus diffusibility. (perezgutierrez2023biologyandtherapeutic pages 1-3, perezgutierrez2023biologyandtherapeutic pages 5-6)

• VEGF121 is described as freely diffusible. (perezgutierrez2023biologyandtherapeutic pages 5-6)
• VEGF165 is partly diffusible and partly HSPG-bound, and ~half of secreted VEGF165 is diffusible while the remainder is HSPG-bound. (perezgutierrez2023biologyandtherapeutic pages 1-3)
• Longer isoforms (e.g., VEGF189/206) are largely sequestered in the ECM via heparin-binding regions. (perezgutierrez2023biologyandtherapeutic pages 5-6)

Proteolytic processing (shorter bioactive forms): VEGFA isoforms can be proteolytically processed into shorter fragments (for example VEGF110/VEGF113), contributing to control of VEGFA bioavailability and vascular patterning. (perezgutierrez2023biologyandtherapeutic pages 1-3, perezgutierrez2023biologyandtherapeutic pages 17-17)

Alternative splice-forms with debated inhibitory biology: So-called VEGFxxxb transcripts (e.g., VEGF165b) have been discussed as anti-angiogenic competitors, but an authoritative 2023 review emphasizes that such variants are better regarded as weak agonists rather than antagonists, and their endogenous existence/importance remains debated. (perezgutierrez2023biologyandtherapeutic pages 15-16, perezgutierrez2023biologyandtherapeutic pages 17-17)

Non-canonical translation and proteolytic processing (L-VEGF → N-VEGF + VEGF-A): Under hypoxia, a long VEGF-A isoform (“L-VEGF”) can be translated from a non-canonical upstream CUG start codon, adding an N-terminal extension. L-VEGF can then be proteolytically cleaved upstream of the canonical VEGF-A start to produce two products: (i) secreted VEGF-A, and (ii) an N-terminal fragment “N-VEGF” that can translocate to the nucleus. (katsman2022nvegftheautoregulatory pages 1-3, katsman2022nvegftheautoregulatory pages 10-13)

1.3 Receptors/co-receptors and downstream signaling pathways

Receptors/co-receptors: VEGF-A binds VEGF receptors VEGFR1 and VEGFR2 and co-receptors neuropilin-1 (NRP1) and neuropilin-2 (NRP2). Ligand binding triggers receptor dimerization and transphosphorylation. (perezgutierrez2023biologyandtherapeutic pages 5-6)

VEGFR2 (KDR/FLK1) signaling outputs: VEGFR2 activation engages multiple canonical pathways:
- PLCγ → PKC → ERK1/2, associated with endothelial proliferation, migration, and broader endothelial homeostasis programs (including pro-angiogenic responses). (perezgutierrez2023biologyandtherapeutic pages 5-6)
- PI3K → AKT, supporting survival and contributing to permeability regulation. (perezgutierrez2023biologyandtherapeutic pages 5-6)
- FAK signaling, linked to ECM adhesion and vascular permeability. (perezgutierrez2023biologyandtherapeutic pages 5-6)

VEGFR1 (FLT1) as regulator/decoy: VEGFR1 can act as a decoy receptor limiting the amount of VEGF-A available to activate VEGFR2 in physiological contexts, and is generally characterized by weaker downstream activation (for example limited PLCγ–PKC–MAPK activation). (li2025advancesinthe pages 1-2, perezgutierrez2023biologyandtherapeutic pages 5-6)

1.4 Regulation of VEGFA expression (hypoxia/HIF axis)

Hypoxia-dependent transcription: VEGFA is a canonical hypoxia-inducible gene regulated by HIF1α/HIF2α binding to a conserved hypoxia response element, and constitutive HIF stabilization (e.g., via loss of VHL function) can drive VEGFA upregulation even in normoxia. (perezgutierrez2023biologyandtherapeutic pages 5-6)

HIF-independent mechanisms: Hypoxic regulation can also occur through HIF-independent routes (for example KRAS-mediated mechanisms), and PI3K/AKT is implicated in hypoxia-induced VEGF expression in some contexts. (perezgutierrez2023biologyandtherapeutic pages 5-6)

2) Recent developments and latest research (prioritizing 2023–2024)

2.1 2023 authoritative synthesis: VEGFA isoform complexity + proteolysis + translation readthrough debates

A highly cited 2023 Nature Reviews Molecular Cell Biology review emphasizes that VEGFA signaling should be understood as a system controlled by alternative splicing, proteolysis, and additional mechanisms such as translational readthrough, and that isoform/processing diversity shapes VEGF potency, ECM interactions, and spatial patterning. (perezgutierrez2023biologyandtherapeutic pages 17-17, perezgutierrez2023biologyandtherapeutic pages 1-3)

This review also provides an expert perspective that proposed anti-angiogenic VEGFA splice variants (VEGFxxxb) are more consistent with weak agonism than antagonism and highlights ongoing debate around endogenous inhibitory isoforms, which is important when interpreting isoform-specific experiments or proposed isoform-specific therapeutics. (perezgutierrez2023biologyandtherapeutic pages 15-16, perezgutierrez2023biologyandtherapeutic pages 17-17)

2.2 2024 real-world pharmacovigilance: ocular adverse event reporting patterns for anti-VEGF drugs

A 2024 analysis using WHO’s VigiAccess/VigiBase spontaneous reporting data (retrieved through Jan 28, 2024) compiled 57,779 adverse-event reports for four commonly used anti-VEGF drugs for wet AMD: ranibizumab (24,338), aflibercept (28,524), brolucizumab (4,065), faricimab (852). (li2024arealworlddata pages 2-4)

The most common system-organ-class categories were eye disorders (43.56%), general disorders/administration site conditions (34.47%), injury/poisoning/procedural complications (13.36%), infections/infestations (11.61%), and nervous system disorders (9.99%), and reports were disproportionately recorded in older patients (highest in >75 years) and more frequently in females (67.83%). (li2024arealworlddata pages 1-2, li2024arealworlddata pages 4-5)

Interpretation note: These are spontaneous reporting volumes, not incidence rates, and are influenced by drug market duration and reporting behaviors. (li2024arealworlddata pages 2-4)

2.3 Emerging durable suppression strategies: gene therapy encoding anti-VEGF biologics (ClinicalTrials.gov)

A major translational development is AAV-based ocular gene therapy aiming to convert the eye into a long-term producer of anti-VEGF biologics, potentially reducing injection burden.

ClinicalTrials.gov records describe ABBV-RGX-314 (RGX-314; surabgene lomparvovec) as an AAV8 vector encoding an anti-VEGF Fab intended to provide sustained intraocular anti-VEGF expression after a single administration. (NCT03066258 chunk 1, NCT04704921 chunk 1)

Key registered trials include:
- NCT03066258 (Phase 1/2a; subretinal; completed; enrolled ~42) dose-escalation with safety focus and follow-up to 104 weeks. (NCT03066258 chunk 1)
- NCT04514653 (Phase 2; suprachoroidal; active not recruiting; enrolled 146) randomized dose-escalation versus ranibizumab, with BCVA and safety endpoints. (NCT04514653 chunk 1)
- NCT04704921 (Phase 2b/3 pivotal; subretinal; active not recruiting; enrolled 671) comparing two RGX-314 dose levels to monthly ranibizumab; primary endpoint BCVA change at Week 54. (NCT04704921 chunk 1)
- NCT05407636 (Phase 3; subretinal; recruiting; planned enrollment 714) comparing two RGX-314 dose levels to aflibercept; primary endpoint BCVA change at Week 54. (NCT05407636 chunk 1)

3) Localization: where VEGFA functions in/around the cell

3.1 Secreted VEGF-A: extracellular ligand with isoform-dependent matrix binding

Canonical VEGF-A isoforms are secreted and act predominantly in the extracellular space to activate VEGFRs on endothelial cells. A key localization principle is bioavailability:
- Some isoforms (e.g., VEGF121) are more diffusible.
- Others (e.g., VEGF189/206) are strongly ECM/HSPG-associated.
This shapes signaling gradients and patterning in tissues. (perezgutierrez2023biologyandtherapeutic pages 5-6, perezgutierrez2023biologyandtherapeutic pages 1-3)

3.2 L-VEGF/N-VEGF: intracellular processing and nuclear function

In the L-VEGF axis, VEGFA biology includes an intracellular/nuclear arm:
- L-VEGF is generated via non-canonical CUG translation under hypoxia and cleaved into secreted VEGF plus N-VEGF. (katsman2022nvegftheautoregulatory pages 1-3)
- N-VEGF is retained intracellularly and can translocate to the nucleus. (katsman2022nvegftheautoregulatory pages 1-3)

Primary experimental evidence shows that forcing N-VEGF into the nucleus (e.g., via NLS fusion) is sufficient to induce transcriptional programs including Hif1α and VEGF-A isoforms under normoxia, and that genetic deletion of N-VEGF changes a substantial fraction of the hypoxia-responsive transcriptome and increases susceptibility to hypoxia-associated apoptosis. (katsman2022nvegftheautoregulatory pages 6-7, katsman2022nvegftheautoregulatory pages 7-10)

4) Pathway context: biological processes and expert mechanistic synthesis

4.1 Angiogenesis, endothelial survival, and permeability as primary pathway-level roles

VEGF-A–VEGFR2 signaling integrates multiple endothelial functions, including proliferation/migration (PLCγ–PKC–ERK), survival (PI3K–AKT), and adhesion/permeability (FAK-related signaling), providing a mechanistic explanation for why VEGFA is both a developmental angiogenesis factor and a mediator of pathological neovascularization and edema. (perezgutierrez2023biologyandtherapeutic pages 5-6)

4.2 Expert opinion (authoritative review perspective)

The 2023 expert review by Pérez-Gutiérrez and Ferrara frames VEGFA as a pathway essential not only for disease angiogenesis but also for adult organ homeostasis, with clinical manipulation requiring careful balancing due to potential harms of VEGF insufficiency or excess. (perezgutierrez2023biologyandtherapeutic pages 15-16)

5) Current applications and real-world implementations

5.1 Ophthalmology: anti-VEGF for neovascular eye diseases

Anti-VEGF therapy has “markedly improved vision” in neovascular eye disease, but practical limitations include short intraocular durability and need for repeated injections, incomplete response in some patients, and procedural risks. (li2025advancesinthe pages 1-2)

Real-world effectiveness and treatment intensity (injection frequency and outcomes): Registry-scale analyses of routine practice demonstrate lower gains than randomized trials, consistent with undertreatment/discontinuation patterns.

A large IRIS-based real-world dataset summarized in a 2025 analysis reports typical injection frequencies around 5.6 (year 1), 3.4 (year 2), 3.1 (year 3), with mean BCVA change of only +0.7 letters at year 1 and decline by −3.1 letters by year 3, and >1/3 discontinuing by year 3. (gyenes2025evaluationofthe pages 7-9)

In a six-year IRIS cohort (reported as 254,655 eyes), mean BCVA changed +3.0 letters at year 1 but net −4.6 letters at 6 years; injections averaged 7.2 (year 1), 5.6 (year 2), and ~4.2–4.6 per year in years 3–6. In that cohort 38.8% had treatment cessation and 32.3% changed treatment; 58.5% lost ≥10 letters at least once, and 14.5% had sustained poor vision after a median 3.4 years. (gyenes2025evaluationofthe pages 7-9)

Discontinuation in multi-center practice: In an Italian multi-center cohort of 2,302 treatment-naïve nAMD patients starting therapy 2019–2021, 28.5% (655) discontinued, with the most common discontinuation category being clinical decision (with subcategories including no signs of exudation and poor/non-response), followed by continuation elsewhere, death, and patient choice. (borrelli2025incidenceandreasons pages 1-2, borrelli2025incidenceandreasons pages 2-3)

5.2 Safety surveillance (real-world pharmacovigilance)

WHO-VigiAccess reporting for four anti-VEGF agents totaled 57,779 reports as of 2023, and the largest category was eye disorders (43.56%). (li2024arealworlddata pages 4-5)

5.3 Durable/one-time approaches: RGX-314 gene therapy (real-world implementation in development)

RGX-314/ABBV-RGX-314 is in late-stage clinical development with both subretinal and suprachoroidal delivery strategies, testing whether a one-time AAV8 gene therapy encoding an anti-VEGF Fab can maintain vision while reducing supplemental injection burden versus standard intravitreal anti-VEGF regimens. (NCT04514653 chunk 1, NCT04704921 chunk 1, NCT05407636 chunk 1)

6) Summary table (molecular forms → function → translation)

Table: This table summarizes the main functional VEGFA products relevant to human UniProt P15692, including splice isoforms, proteolytic fragments, and the long-form L-VEGF/N-VEGF pathway. It also links these molecular forms to receptor signaling and current clinical translation in ophthalmology and oncology.

7) Key takeaways for functional annotation of VEGFA (UniProt P15692)

1. Primary molecular function: VEGF-A is a secreted signaling ligand whose main biochemical role is receptor binding/activation (VEGFR1/VEGFR2 with neuropilin co-receptors), driving endothelial signaling through PLCγ–PKC–ERK, PI3K–AKT, and FAK-associated pathways to control angiogenesis, survival, and permeability. (perezgutierrez2023biologyandtherapeutic pages 5-6)
2. Form-dependent bioavailability is central: Alternative splicing and matrix binding (HSPG/ECM retention) are not peripheral details; they are core determinants of VEGFA spatial signaling and thus of phenotype. (perezgutierrez2023biologyandtherapeutic pages 1-3, perezgutierrez2023biologyandtherapeutic pages 5-6)
3. VEGFA includes an intracrine/nuclear arm: The L-VEGF → N-VEGF axis implies VEGFA can contribute directly to hypoxia adaptation through a nuclear fragment that reshapes gene expression and cell survival under hypoxic stress. (katsman2022nvegftheautoregulatory pages 1-3, katsman2022nvegftheautoregulatory pages 7-10)
4. Clinical translation is mature yet evolving: Anti-VEGF therapies are foundational in ophthalmology, but real-world data show injection burden, discontinuation, and suboptimal long-term outcomes; durable approaches including gene therapy (RGX-314) represent a major active implementation pathway. (gyenes2025evaluationofthe pages 7-9, borrelli2025incidenceandreasons pages 1-2, NCT04514653 chunk 1)

Source details (URLs and publication dates)

• Pérez-Gutiérrez L, Ferrara N. Biology and therapeutic targeting of vascular endothelial growth factor A. Nat Rev Mol Cell Biol. Jul 2023. https://doi.org/10.1038/s41580-023-00631-w (perezgutierrez2023biologyandtherapeutic pages 1-3)
• Li C, Lu Y, Song Z, Liu Y. A real-world data analysis of ocular adverse events linked to anti-VEGF drugs: a WHO-VigiAccess study. Front Pharmacol. Jul 2024. https://doi.org/10.3389/fphar.2024.1398783 (li2024arealworlddata pages 1-2)
• Katsman M, et al. N-VEGF, the autoregulatory arm of VEGF-A. Cells. Mar 2022. https://doi.org/10.3390/cells11081289 (katsman2022nvegftheautoregulatory pages 1-3)
• ClinicalTrials.gov: NCT03066258 (posted/updated metadata available on record; gene therapy RGX-314; subretinal; completed). https://clinicaltrials.gov/study/NCT03066258 (NCT03066258 chunk 1)
• ClinicalTrials.gov: NCT04514653 (AAVIATE; suprachoroidal ABBV-RGX-314; active not recruiting). https://clinicaltrials.gov/study/NCT04514653 (NCT04514653 chunk 1)
• ClinicalTrials.gov: NCT04704921 (ATMOSPHERE; subretinal ABBV-RGX-314; active not recruiting). https://clinicaltrials.gov/study/NCT04704921 (NCT04704921 chunk 1)
• ClinicalTrials.gov: NCT05407636 (Phase 3; subretinal ABBV-RGX-314; recruiting). https://clinicaltrials.gov/study/NCT05407636 (NCT05407636 chunk 1)

References

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7. (li2025advancesinthe pages 1-2): Huan Li and Xiong Huang. Advances in the molecular signaling mechanisms of vegf/vegfr2 in fundus neovascularization disease (review). Experimental and Therapeutic Medicine, 30:1-13, May 2025. URL: https://doi.org/10.3892/etm.2025.12893, doi:10.3892/etm.2025.12893. This article has 7 citations and is from a peer-reviewed journal.

8. (li2024arealworlddata pages 2-4): Chen Li, Yicheng Lu, Ziyue Song, and Yueqi Liu. A real-world data analysis of ocular adverse events linked to anti-vegf drugs: a who-vigiaccess study. Frontiers in Pharmacology, Jul 2024. URL: https://doi.org/10.3389/fphar.2024.1398783, doi:10.3389/fphar.2024.1398783. This article has 7 citations.

9. (li2024arealworlddata pages 1-2): Chen Li, Yicheng Lu, Ziyue Song, and Yueqi Liu. A real-world data analysis of ocular adverse events linked to anti-vegf drugs: a who-vigiaccess study. Frontiers in Pharmacology, Jul 2024. URL: https://doi.org/10.3389/fphar.2024.1398783, doi:10.3389/fphar.2024.1398783. This article has 7 citations.

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11. (NCT03066258 chunk 1): Safety and Tolerability of RGX-314 (Investigational Product) Gene Therapy for Neovascular AMD Trial. REGENXBIO Inc.. 2017. ClinicalTrials.gov Identifier: NCT03066258

12. (NCT04704921 chunk 1): Pivotal 1 Study of ABBV-RGX-314 (Also Known as RGX-314) Gene Therapy Administered Via Subretinal Delivery One Time in Participants With nAMD. AbbVie. 2020. ClinicalTrials.gov Identifier: NCT04704921

13. (NCT04514653 chunk 1): RGX-314 Gene Therapy Administered in the Suprachoroidal Space for Participants With Neovascular Age-Related Macular Degeneration (nAMD). AbbVie. 2020. ClinicalTrials.gov Identifier: NCT04514653

14. (NCT05407636 chunk 1): Pivotal 2 Study of RGX-314 Gene Therapy in Participants With nAMD. AbbVie. 2022. ClinicalTrials.gov Identifier: NCT05407636

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19. (borrelli2025incidenceandreasons pages 2-3): Enrico Borrelli, Claudio Foti, Lorena Ulla, Annamaria Porreca, Ugo Introini, Maria Oliva Grassi, Pasquale Viggiano, Mario Peronetti, Rebecca Toscani, Giacomo Boscia, Alba Chiara Termite, Carlo Gennaro, Paola Marolo, Francesco Boscia, Francesco Bandello, and Michele Reibaldi. Incidence and reasons for discontinuation of anti-vegf treatment in neovascular age-related macular degeneration. The British Journal of Ophthalmology, 109:875-881, Mar 2025. URL: https://doi.org/10.1136/bjo-2024-326152, doi:10.1136/bjo-2024-326152. This article has 10 citations.

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21. (lee2025vascularendothelialgrowth pages 1-2): Chunsik Lee, Myung-Jin Kim, Anil Kumar, Han-Woong Lee, Yunlong Yang, and Yonghwan Kim. Vascular endothelial growth factor signaling in health and disease: from molecular mechanisms to therapeutic perspectives. Signal Transduction and Targeted Therapy, May 2025. URL: https://doi.org/10.1038/s41392-025-02249-0, doi:10.1038/s41392-025-02249-0. This article has 294 citations and is from a peer-reviewed journal.

22. (li2025advancesinthe pages 13-13): Huan Li and Xiong Huang. Advances in the molecular signaling mechanisms of vegf/vegfr2 in fundus neovascularization disease (review). Experimental and Therapeutic Medicine, 30:1-13, May 2025. URL: https://doi.org/10.3892/etm.2025.12893, doi:10.3892/etm.2025.12893. This article has 7 citations and is from a peer-reviewed journal.

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Citations

1. perezgutierrez2023biologyandtherapeutic pages 5-6
2. perezgutierrez2023biologyandtherapeutic pages 1-3
3. li2024arealworlddata pages 2-4
4. katsman2022nvegftheautoregulatory pages 1-3
5. perezgutierrez2023biologyandtherapeutic pages 15-16
6. li2025advancesinthe pages 1-2
7. gyenes2025evaluationofthe pages 7-9
8. li2024arealworlddata pages 4-5
9. li2024arealworlddata pages 1-2
10. perezgutierrez2023biologyandtherapeutic pages 17-17
11. katsman2022nvegftheautoregulatory pages 10-13
12. katsman2022nvegftheautoregulatory pages 6-7
13. katsman2022nvegftheautoregulatory pages 7-10
14. borrelli2025incidenceandreasons pages 1-2
15. borrelli2025incidenceandreasons pages 2-3
16. lee2025vascularendothelialgrowth pages 3-4
17. lee2025vascularendothelialgrowth pages 1-2
18. li2025advancesinthe pages 13-13
19. https://doi.org/10.1038/s41580-023-00631-w
20. https://doi.org/10.3389/fphar.2024.1398783
21. https://doi.org/10.3390/cells11081289
22. https://clinicaltrials.gov/study/NCT03066258
23. https://clinicaltrials.gov/study/NCT04514653
24. https://clinicaltrials.gov/study/NCT04704921
25. https://clinicaltrials.gov/study/NCT05407636
26. https://doi.org/10.1038/s41580-023-00631-w,
27. https://doi.org/10.3390/cells11081289,
28. https://doi.org/10.3892/etm.2025.12893,
29. https://doi.org/10.3389/fphar.2024.1398783,
30. https://doi.org/10.3390/ijms262210927,
31. https://doi.org/10.1136/bjo-2024-326152,
32. https://doi.org/10.1038/s41392-025-02249-0,

Comprehensive Functional Annotation of VEGFA (Vascular Endothelial Growth Factor A)

VEGFA, located on human chromosome 6p21.1, encodes vascular endothelial growth factor A, a critical secreted signaling protein that serves as the master regulator of angiogenesis and vasculogenesis across virtually all physiological and pathological contexts.[1][3] This dimeric growth factor exists as multiple isoforms generated through alternative splicing, ranging from 121 to 206 amino acids in length, each conferring distinct bioavailability and biological potency through differential interactions with heparan sulfate proteoglycans and the extracellular matrix.[2][7][32] VEGFA functions primarily as a ligand for two receptor tyrosine kinases, VEGFR1 and VEGFR2, though it also engages the non-kinase co-receptor neuropilin-1, thereby orchestrating complex intracellular signaling cascades that promote endothelial cell proliferation, survival, migration, and increased vascular permeability.[3][4][13] The protein contains a characteristic cystine-knot motif stabilized by multiple interchain and intrachain disulfide bonds, conferring remarkable structural stability even under hypoxic conditions where oxygen availability is severely compromised.[11][19] Beyond its well-established role as a paracrine angiogenic factor, VEGFA is increasingly recognized as functioning in intracrine signaling pathways within multiple cell types including cancer cells, hematopoietic stem cells, and various macrophage populations, where nuclear-localized VEGFA and its receptors directly regulate gene expression, cell survival, and metabolic homeostasis.[48] This multifaceted protein exemplifies how alternative splicing and spatial compartmentalization of signaling create a single gene product capable of executing distinct biological functions appropriate to specific developmental, regenerative, and pathological contexts.

Gene Structure and Chromosomal Organization

The VEGFA gene spans a 16.3 kilobase coding region located at chromosome 6p21.1 on the short arm of the human genome, organized into eight exons separated by seven introns.[43] This compact genomic organization belies the remarkable diversity of VEGFA protein isoforms that can be generated from the single gene through multiple mechanisms of alternative splicing. The gene structure reflects evolutionary conservation of the PDGF/VEGF growth factor family, with sequence homology particularly evident in the regions encoding the critical cystine-knot motif and receptor-binding domains.[1] The location on chromosome 6 places VEGFA within a gene-rich region, and the genomic sequence has been characterized at high resolution through multiple reference genome assemblies including GRCh38.p14 and alternate T2T-CHM13v2.0 assemblies.[1] Beyond the constitutive elements of the gene, the regulatory landscape encompasses promoter regions responsive to multiple environmental signals including hypoxia, inflammatory cytokines, and growth factors, ensuring that VEGFA expression can be rapidly induced or suppressed in response to tissue-specific demands. The identification of multiple single nucleotide polymorphisms (SNPs) within the VEGFA locus has revealed significant associations with circulating VEGF-A levels and cardiovascular disease risk, indicating that genetic variation at this locus modulates both baseline VEGF expression and individual susceptibility to vascular disorders.[43]

Protein Structure and Cystine-Knot Motif

The VEGFA monomer adopts a distinctive three-dimensional structure anchored by a cystine-knot motif, a structural feature shared with other members of the PDGF/VEGF cytokine superfamily including platelet-derived growth factor and transforming growth factor beta.[22][31] The cystine knot consists of three disulfide bonds arranged in a topologically unique manner: the first two disulfide bridges form an eight-residue ring, through which a third disulfide bond passes, creating the characteristic knot-like topology.[22][31] Specifically, in VEGF these disulfide bonds occur between cysteines at positions 57-102, 61-104, and 26-68, with the latter passing through the ring formed by the first two.[22] This structural motif provides exceptional stability to the VEGF monomer and is essential for subsequent dimerization and receptor engagement. The VEGFA protein forms antiparallel homodimers covalently linked by two additional interchain disulfide bridges between cysteine residues 51 and 60 of each monomer, creating a molecule with approximate molecular weight of 46 kilodaltons.[22][31] Around this cystine-knot core, the VEGFA monomer contains a central four-stranded beta sheet extending from residues that form part of the receptor-binding interface.[22] The monomer structure includes an N-terminal alpha helix, which distinguishes VEGFA from PDGF and contributes to the larger buried surface area at the dimer interface compared to other cystine-knot family members.[22] This structural organization creates two symmetrical receptor-binding sites located at opposite poles of the homodimer, allowing simultaneous engagement of two VEGFR dimers and enabling the formation of higher-order signaling complexes.[22][31]

The structural stability conferred by the cystine-knot motif proves particularly important for VEGFA function under hypoxic conditions. Recent evidence demonstrates that VEGFA can efficiently complete disulfide bond formation and folding in the endoplasmic reticulum even in the absence of sufficient oxygen, unlike many other growth factors and structural proteins that become misfolded under hypoxia.[19] This oxygen-independent disulfide bond formation capability reflects selective evolutionary pressure on hypoxia-induced proteins to maintain functionality when oxygen is severely limited, suggesting that the compact disulfide architecture of VEGFA represents an adaptation for expression during the hypoxic conditions that characterize developing tissues and ischemic regions.[19] The protein contains an N-linked glycan as part of its core structure, and additional post-translational modifications including glycosylation at multiple sites throughout the molecule are essential for proper folding, secretion, and biological activity.[20] These structural features create a remarkably stable growth factor capable of maintaining signaling function across diverse physiological contexts.

Alternative Splicing and Isoform Diversity

The VEGFA gene encodes a remarkable diversity of protein isoforms through alternative splicing of exons 5, 6, 7, and 8, creating two major families of proteins with profoundly different biological properties.[2][7][57] Differential splicing of exons 6 and 7 generates the main VEGF family of proangiogenic isoforms, which exist as VEGF121, VEGF165, VEGF189, and VEGF206, corresponding to their amino acid lengths.[2][7][57] VEGF121 arises from skipping both exons 6 and 7 and represents the shortest proangiogenic form, existing as a weakly acidic polypeptide that lacks fifteen basic amino acids within the forty-four residues encoded by exon 7.[7][57] This shorter form is freely diffusible and does not bind to heparin or heparan sulfate proteoglycans, resulting in unrestricted diffusion from cells and minimal accumulation in the extracellular matrix.[7][32] In contrast, VEGF165, produced when exon 7 is retained but exon 6 is skipped, possesses a basic cluster of amino acids within its carboxyl terminus encoded by exon 7, conferring intermediate heparin-binding affinity.[7][32] This intermediate property of VEGF165 appears to represent an optimal balance between bioavailability and receptor engagement, as mice expressing only VEGF164 (the mouse ortholog) develop normally, suggesting this isoform achieves the "perfect balance" between diffusibility and heparan sulfate proteoglycan binding.[32]

The longer isoforms VEGF189 and VEGF206 retain exon 6, which encodes a twenty-four amino acid insertion highly enriched in arginine and lysine residues, creating highly basic proteins with extremely high affinity for heparin and heparan sulfate proteoglycans.[7][32] Consequently, VEGF189 and VEGF206 are predominantly sequestered in the extracellular matrix and cell surface rather than secreted into the soluble fraction, functioning as storage forms of VEGF that must be proteolytically processed by plasmin, matrix metalloproteinases, or other proteases to be released as bioactive, freely diffusible fragments.[32] This isoform-specific storage mechanism creates a spatial gradient of VEGF bioavailability, with diffusible shorter isoforms providing dynamic angiogenic signaling while matrix-bound longer isoforms serve as readily mobilizable reserves that can be activated during tissue remodeling or upon demand.[32] The differential expression of VEGF isoforms varies by tissue type and developmental stage, with longer isoforms predominating in certain tissues such as normal lung tissue, while shorter isoforms become relatively enriched in cancer and during pathological angiogenesis.[57]

Beyond the primary family of proangiogenic isoforms, alternative splice-site selection within exon 8 generates an entirely different class of proteins termed VEGF165b, VEGF121b, and related antiangiogenic variants.[2][7][38] Alternative splicing at the 3' end of exon 8 produces two mutually exclusive exon sequences: exon 8a encoding cysteine-aspartate-lysine-proline-arginine-arginine and exon 8b encoding serine-leucine-threonine-arginine-lysine-aspartate.[7] These six C-terminal amino acid differences confer radically different functional properties, with VEGF165b and other VEGFxxxb isoforms demonstrating antiangiogenic activity that opposes the proangiogenic effects of their conventional VEGF165 and VEGFxxx counterparts.[2][38] The antiangiogenic isoforms are expressed at elevated levels in normal, quiescent tissues but become dramatically downregulated in angiogenic diseases including cancer, proliferative retinopathy, and developmental pathologies such as preeclampsia and Denys-Drash syndrome.[2][38] These observations suggest that the balance between pro- and antiangiogenic VEGF isoforms may represent a fundamental regulatory mechanism controlling angiogenic potential, with loss of antiangiogenic isoforms contributing to pathological angiogenesis in disease states.

VEGF Receptor Structure and Binding Interactions

VEGFA exerts its biological effects primarily through two receptor tyrosine kinases designated VEGFR1 (also known as Flt-1 or FMS-like tyrosine kinase 1) and VEGFR2 (also known as KDR or kinase domain region), though it also engages co-receptors including neuropilin-1 and neuropilin-2.[3][6][13] Both VEGFR1 and VEGFR2 are membrane-bound receptor tyrosine kinases belonging to the split kinase domain family of receptors, featuring seven immunoglobulin-like extracellular domains, a single transmembrane spanning region, and an intracellular domain containing a split tyrosine kinase.[6][13][14] The extracellular portions of both receptors, particularly the immunoglobulin-like domains 2 and 3 (IgD2 and IgD3), provide the primary VEGF-binding surfaces.[14][17] Despite structural similarity, VEGFR1 and VEGFR2 differ substantially in their functional consequences when activated by VEGF: VEGFR1 binds VEGF-A with approximately 50-fold higher affinity than VEGFR2, yet exhibits only weak tyrosine phosphorylation in endothelial cells upon VEGF-induced dimerization, resulting in minimal activation of downstream angiogenic signaling.[6][30] In contrast, VEGFR2 demonstrates more robust tyrosine phosphorylation upon VEGF engagement and mediates essentially all of the characterized pro-angiogenic responses to VEGF in endothelial cells, including proliferation, survival, migration, and vascular permeability.[3][6][13][14]

This apparent paradox of higher binding affinity paired with weaker signaling at VEGFR1 is resolved through understanding the distinct biological roles of these receptors: VEGFR1 functions primarily as a negative regulator of VEGFR2 signaling during early vascular development, and can function as a decoy receptor sequestering VEGF from VEGFR2 engagement.[6][30] A soluble splice variant of VEGFR1 designated sFlt-1 (soluble fms-like tyrosine kinase 1) is secreted and functions as a circulating VEGF trap, effectively neutralizing circulating VEGF and preventing its interaction with cell surface VEGFR2.[6][30] This regulatory mechanism becomes physiologically critical during pregnancy, where the balance between circulating VEGF-A and sFlt-1 determines vascular permeability and endothelial function; dysregulation of this balance contributes to preeclampsia pathogenesis.[6] A third VEGF receptor designated VEGFR3 mediates responses to VEGF-C and VEGF-D but does not serve as a VEGF-A ligand, instead functioning in lymphangiogenesis.[6]

The co-receptor neuropilin-1 dramatically enhances VEGF signaling through mechanisms that remained incompletely understood until recent research clarified its role. VEGF165, the predominant isoform, binds to neuropilin-1 through its heparin-binding domain encoded by exon 7, whereas the shorter VEGF121 isoform, lacking exon 7, does not bind neuropilin-1.[8][9][12] The crystal structure of neuropilin-1 b1b2 domains in complex with a VEGF-derived peptide revealed that the basic tail of VEGF165 interacts directly with acidic residues on the neuropilin-1 b1 domain, with heparin serving as a critical bridge molecule that enhances the interaction between VEGF, neuropilin-1, and the extracellular matrix.[8][9] Surprisingly, recent genetic evidence demonstrates that neuropilin-1 promotes angiogenesis not primarily through enhancing VEGF binding, but rather through functioning as a co-receptor that regulates cell surface expression and trafficking of VEGFR2.[12] Specifically, neuropilin-1 guides internalized VEGFR2 from lysosomal degradation pathways into Rab11-positive recycling vesicles, thereby stabilizing VEGFR2 at the cell surface and amplifying VEGFR2-VEGF signaling.[12] This co-receptor function explains how neuropilin-1 can regulate angiogenesis even when direct VEGF-neuropilin-1 binding is disrupted, establishing neuropilin-1 as a critical regulator of endothelial cell responsiveness to VEGF rather than solely a ligand-binding protein.

VEGFR2 Signaling Pathways and Downstream Effectors

Upon VEGF-A binding and VEGFR2 dimerization, the receptor undergoes rapid autophosphorylation at multiple tyrosine residues within its intracellular domains, with each phosphorylation site serving as a docking platform for specific signaling adaptor proteins that initiate distinct downstream cascades.[14][17] The juxtamembrane domain tyrosine Y801 undergoes phosphorylation as part of the activation mechanism, where in its unphosphorylated state this domain physically interacts with the kinase domain activation loop in an autoinhibitory configuration.[14] Phosphorylation at Y801 relieves this autoinhibition, allowing the kinase domain to adopt an active conformation capable of transphosphorylating additional tyrosine residues.[14] Within the kinase domain proper, phosphorylation at Y951 and Y1054/Y1059 further enhance kinase catalytic activity, while the C-terminal domain contains critical sites at Y1175 and Y1214 that recruit specific effector proteins.[14][17]

The phosphorylated Y1175 residue recruits phospholipase C-gamma (PLCγ) through its SH2 domain, initiating the PLCγ-PKC-MAPK pathway that represents the predominant pro-proliferative signal in endothelial cells.[13][14][17][25] PLCγ activation catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), leading to activation of protein kinase C (PKC) and elevation of intracellular calcium concentration.[13][17] PKC then activates the Raf-MEK-ERK mitogen-activated protein kinase cascade, which translocates into the nucleus and activates transcription factors that induce gene expression required for endothelial cell DNA synthesis and proliferation.[13][17] Notably, analysis of VEGF-A isoforms reveals that different isoforms differentially activate this critical Y1175-PLCγ pathway, with VEGF165 inducing much greater ERK1/2 phosphorylation than VEGF121, suggesting that isoform-specific differences in signaling capacity may explain their distinct biological potencies.[2] The differential coupling to this pathway operates through variations in receptor phosphorylation kinetics, with VEGF165 stimulation of endothelial cells producing greater and more sustained VEGFR2 Y1175 phosphorylation compared to VEGF121.[2]

Phosphorylation of VEGFR2 at Y951 within the kinase insert domain initiates the TSAd-Src-PI3K-Akt pathway, which is essential for endothelial cell survival.[13][14][17] Src-family kinases are recruited to phosphorylated Y951 and subsequently activate phosphoinositide 3-kinase (PI3K), which catalyzes the conversion of PIP2 to phosphatidylinositol 3,4,5-trisphosphate (PIP3).[14][17] PIP3 then recruits and activates protein kinase B (Akt/PKB), which phosphorylates and thereby inactivates pro-apoptotic proteins including BAD (BCL2-associated agonist of cell death) and caspase-9, ensuring endothelial cell survival under VEGF stimulation.[14][17] This survival signal proves critical for maintaining vascular integrity and preventing endothelial cell loss under stress conditions. Additionally, Y951-mediated PI3K-Akt activation contributes to vascular permeability through parallel mechanisms including Rac1 GTPase activation and reactive oxygen species production.[33]

Phosphorylation at Y1214 recruits the NCK adaptor protein and initiates signaling through p38 MAPK and downstream MAPKAPK2/3 kinases, leading to actin remodeling and endothelial cell migration essential for angiogenic sprouting.[14][17][51] This pathway also involves activation of small GTPases including Rac and Cdc42, which orchestrate cytoskeletal reorganization required for directed cell movement.[14][51] The Y1175 site also recruits SHB adaptor protein, linking to FAK (focal adhesion kinase) and paxillin through a separate signaling axis that modulates focal adhesion turnover and cellular motility.[14] Collectively, these multiple phosphorylation sites on VEGFR2 enable the single VEGF ligand to activate multiple distinct signaling pathways within a single cell, allowing VEGF to simultaneously promote proliferation, survival, and migration—the three key endothelial functions required for effective angiogenesis.

Recent evidence has identified an additional layer of complexity in VEGFR2 signaling through the discovery that integrin αvβ3 can directly bind the heparin-binding domain of VEGF165, and that this integrin-VEGF interaction regulates VEGFR2 signaling.[35] Specifically, binding sites for integrin αvβ3 and VEGFR2 domain 1 overlap within the heparin-binding domain of VEGF165, suggesting that these two receptors compete for VEGF binding at this site.[35] The functional consequence of αvβ3 binding to VEGF165 is suppression of KDR domain 1 binding and reduced ERK1/2 and Akt phosphorylation, indicating that integrin αvβ3 can function as a negative regulator of VEGF signaling through competitive receptor binding.[35]

Biological Functions in Angiogenesis and Vasculogenesis

VEGF-A functions as the master regulator of angiogenesis, the process of new blood vessel formation from pre-existing vasculature, and vasculogenesis, the de novo formation of blood vessels from vascular progenitor cells, across all developmental and adult contexts.[52] During embryonic development, VEGF is initially expressed at high levels in the yolk sac and at embryonic sites of vessel formation, supporting the assembly of the cardiovascular system beginning around embryonic day 7.5 to 8.0 in mice.[49][52] The spatial localization of VEGF expression in the anterior mesoderm and its co-localization with VEGFR-expressing cells in posterior regions establishes a chemotactic gradient guiding vascular progenitor cell migration from posterior developmental regions toward anterior embryonic structures including the developing heart and forebrain.[49] This gradient-dependent cell migration represents a critical early step in cardiovascular morphogenesis, as inhibition of VEGFR signaling during this window substantially impairs the accumulation and migration of VEGFR-positive cells to their destination tissues.[49]

The process of vasculogenic tubulogenesis—the transformation of solid aggregates of endothelial progenitors (angioblasts) into patent blood vessels—depends absolutely on VEGFR2 signaling, as demonstrated by the embryonic lethality of VEGFR2 null mice at approximately embryonic day 8.5.[52] The development of a functional vascular network proceeds through distinct stages, beginning with the formation of primitive vascular plexuses that subsequently undergo sprouting angiogenesis and vascular remodeling to establish a hierarchical organization of vessels of varying diameters and functions.[44] VEGF initiates sprouting angiogenesis through multiple mechanisms: it stimulates endothelial cell proliferation through the PLCγ-PKC-MAPK pathway, promotes directed migration of endothelial cells through Rac1 and Cdc42-mediated actin remodeling, and increases vascular permeability to facilitate plasma protein extravasation and infiltration of supporting cells.[3][4][51] The temporal progression of VEGF expression during wound healing illustrates this multifaceted role: VEGF levels are dramatically upregulated 3-7 days post-wounding, the proliferative phase of repair during which capillary growth and differentiation peak, and VEGF neutralization with blocking antibodies substantially diminishes both chemotactic and angiogenic properties of wound fluid.[58]

The formation of vascular lumens, the hollow centers through which blood flows, involves specialized VEGF-mediated signaling and endothelial cell polarization mechanisms. During lumen formation, VEGF-activated endothelial cells must undergo dramatic morphological changes including actin remodeling, vesicle-mediated trafficking to the luminal membrane, and junctional reorganization to establish the inside versus outside orientation of developing vessels.[44] This process appears to involve multiple mechanisms coordinating simultaneously: Rab7-directed vectorial transport of vesicles to expand the developing lumen at the cord center, and concurrent redistribution of junctional proteins away from the cord center to preserve peripheral cell-cell contacts while creating a hollow channel.[44] The importance of endothelial cell polarity is evidenced by studies showing that loss of β1 integrin, which disrupts Par3 localization and endothelial polarity, results in accumulation of cytosolic Rab7+ vesicles and failure of vascular lumen formation despite apparently normal early vasculogenesis.[44]

VEGF Secretion, Trafficking, and Subcellular Localization

VEGF-A contains an N-terminal signal sequence targeting it to the lumen of the endoplasmic reticulum, where the nascent protein undergoes rapid dimerization through disulfide bond formation and co-translational oxidation.[20] The trafficking of VEGF from the endoplasmic reticulum through the secretory pathway to the cell surface requires specific small GTPases: Sar1 and Arf1 regulate VEGF165 movement between the endoplasmic reticulum and Golgi apparatus, while post-Golgi transport depends critically on N-linked glycosylation, a modification occurring within the Golgi compartment.[20] These early secretory steps can be regulated by signaling pathways; phorbol ester-induced protein kinase C activation, for instance, promotes VEGF165 secretion by increasing the flux through the entire secretory pathway, while the effect appears sensitive to intracellular calcium elevation.[20] A striking feature of VEGF165 trafficking is that a substantial fraction of the protein localizes to the outer surface of the plasma membrane in a punctate, string-like pattern rather than being immediately secreted into the surrounding medium.[20] From this plasma membrane location, approximately 50% of VEGF165-GFP undergoes shedding from the cell surface in association with membrane vesicles and other membrane components, suggesting a mechanism for regulated release of VEGF into the immediate microenvironment.[20]

Beneath the well-characterized extracellular functions of VEGF lies an emerging role for intracellular VEGF signaling termed intracrine signaling, wherein VEGF functions as a signaling molecule within cells without prior secretion.[48] Nuclear-localized VEGF and VEGFR2 have been detected in multiple cell types including endothelial cells, cancer cells, and hematopoietic cells, and evidence suggests these intracellular receptors participate in direct regulation of nuclear gene expression and metabolic homeostasis.[48] In cultured endothelial cells, VEGF knockdown results not in classical markers of apoptosis such as cleaved caspase-3, but rather in dramatic mitochondrial fragmentation accompanied by reduced metabolic activity and depressed mitochondrial function, suggesting intracrine VEGF maintains normal cellular metabolism through mitochondrial regulation.[48] In cancer cells including acute myeloid leukemia cells, a pool of VEGFR1 localizes to the nucleus rather than the cell surface, and neutralizing anti-VEGF antibodies reduce nuclear VEGFR2 localization while shifting it toward the cell surface, indicating that autocrine VEGF released within cells promotes nuclear localization of its receptors.[48] The specific mechanisms by which nuclear-localized VEGF and VEGFR regulate transcription remain to be fully elucidated, but evidence suggests non-kinase functions of intracellular VEGFR1 may be involved, as some effects cannot be blocked by tyrosine kinase inhibitors.[48] These intracrine functions expand the biological scope of VEGF beyond its classical role as a paracrine angiogenic factor, establishing it as a regulator of fundamental cellular processes including energy metabolism and survival.

Regulation of VEGF Expression

VEGF-A gene expression is regulated by multiple transcriptional and post-transcriptional mechanisms responsive to physiological and pathological signals. Hypoxia represents perhaps the most important physiological regulator of VEGF expression, acting through the hypoxia-inducible factor-1 (HIF-1) pathway wherein low oxygen tension stabilizes HIF-1α protein, which heterodimerizes with HIF-1β and directly binds to hypoxia response elements in the VEGFA promoter region.[21] However, VEGF regulation in certain contexts proceeds through HIF-1-independent pathways, as evidenced by studies showing that in hepatocellular carcinoma cells, VEGF expression remains substantially elevated even when HIF-1α is genetically suppressed, with this HIF-1-independent expression controlled by phosphoinositide 3-kinase and transcription factor SP1.[21] This pathway diversity ensures that VEGF can be rapidly mobilized under conditions of metabolic stress regardless of the specific mechanism triggering cellular adaptation.

Genetic variation at the VEGFA locus, particularly single nucleotide polymorphisms in the gene sequence or regulatory regions, influences baseline VEGF-A levels and individual susceptibility to cardiovascular disease and other vascular disorders.[43] Multiple VEGFA SNPs including rs6921438, rs4416670, rs6993770, and rs10738760 distributed across three chromosomes have been associated with circulating VEGF-A levels, collectively explaining up to 52% of the variance in total circulating VEGF-A.[43] The rs2010963 SNP in exon 1 of VEGFA associates with differential cardiovascular outcomes in different populations, with the CC/GC genotypes conferring increased risk of cardiac death within a five-year follow-up period in some coronary artery disease cohorts.[43] These genetic associations underscore how VEGF-A levels and signaling capacity are subject to substantial individual genetic variation, potentially explaining differential responsiveness to anti-VEGF therapeutics and variable disease progression among individuals exposed to similar risk factors.

At the post-transcriptional level, VEGF-A mRNA undergoes polyadenylation and export from the nucleus, and stability of VEGF mRNA is modulated by microRNA interactions and RNA-binding proteins.[21] The VEGF gene also contains a precursor form arising from initiation at an upstream in-frame CUG codon rather than the standard AUG initiation codon, generating a 47 kilodalton VEGF165 precursor with a 180-residue N-terminal extension.[15] This precursor form is processed by proteolytic cleavage and appears to have different properties than mature VEGF, as the precursor does not induce endothelial cell proliferation or expression of angiopoietin-2, yet adheres to the extracellular matrix, suggesting potential roles as a storage form or for localized VEGF generation.[15]