Recombinant Human Vascular endothelial growth factor A protein (VEGFA), partial (Active)

What is VEGFA and what are its primary biological functions?

VEGFA belongs to the vascular endothelial growth factor family and serves as a potent mediator of angiogenesis. It plays fundamental roles in cell survival and proliferation, particularly in endothelial cells . Originally identified as both a vascular permeability factor (VPF) and tumor angiogenic factor (TAF), VEGFA demonstrates vascular selectivity with high-affinity binding to endothelial cells .

VEGFA functions extend beyond angiogenesis to include:

• Promotion of endothelial cell migration and proliferation

• Enhancement of vascular permeability (50,000 times more potent than histamine on a molar basis)

• Induction of blood vessel growth in various tissue contexts

• Regulation of energy metabolism in certain contexts

• Maintenance of specialized vascular structures such as glomerular filtration barriers

These diverse functions explain why VEGFA has been implicated in numerous physiological and pathological processes including tumor growth, diabetic proliferative retinopathy, inflammatory skin diseases, pulmonary diseases, myocardial infarction, and peripheral vascular disease .

How does VEGFA signaling operate at the cellular level?

VEGFA signals primarily through binding to specific receptors (VEGFR1/FLT1 and VEGFR2/KDR/FLK1) on endothelial cells. The threshold and timing of VEGFR2 activation depend critically on signal strength over a remarkable 50-fold concentration range, with cells capable of distinguishing even 3-fold differences in concentration .

This signaling demonstrates several key characteristics:

• Signal intensity is not interpreted equivalently to signal duration, meaning longer exposure cannot compensate for low concentration

• VEGFA activates multiple downstream pathways, with the PI3K/AKT pathway being particularly important for regenerative processes

• In certain cellular contexts, VEGFA can activate AMPKα (T172 phosphorylation) in a dose-dependent manner, reaching peak activation at higher concentrations (200 nM)

This nuanced signaling pattern enables cells to interpret VEGFA gradients precisely during development and tissue maintenance, critical for understanding experimental outcomes when working with recombinant VEGFA.

What is known about VEGFA genetic polymorphisms and their research implications?

Multiple VEGFA polymorphisms have been identified and studied in relation to disease susceptibility and progression. The most commonly investigated include rs699947 (–1540C>A), rs833061 (–460T>C), rs2010963 (405C>G), rs25648 (1032C>T) and rs3025039 (1689C>T) .

These polymorphisms demonstrate:

• High linkage disequilibrium (LD) between neighboring loci, particularly rs699947, rs833061, rs2010963, and rs25648

• Three main haplotype groups in populations (based on rs699947, rs833061, and rs2010963): ACG (~0.39), CTC (~0.38), and CTG (~0.21)

When designing genetic association studies involving VEGFA, researchers should consider these polymorphisms and their linkage patterns. Genotyping can be performed efficiently using TaqMan MGB probes with validation through DNA sequencing . Haplotype analysis frequently provides more meaningful insights than single SNP analysis due to the high LD between these polymorphisms.

What are optimal methods for producing recombinant human VEGFA protein?

Recombinant VEGFA production typically involves bacterial expression systems with polyhistidine (6×His) tags to facilitate purification. Based on experimental evidence, the following approach has proven effective:

• Clone the VEGFA coding sequence into an appropriate expression vector

• Transform into bacterial expression hosts

• Induce expression with IPTG (optimally 1 mM for 8 hours at 37°C, which yields higher expression than shorter induction periods)

• Extract protein (note that recombinant VEGFA appears in both pellet and supernatant fractions)

• Purify using Ni⁺-NTA affinity columns, leveraging the His-tag

• Confirm identity via Western blot using anti-His antibodies

This approach produces functional VEGFA protein that can be used for various experimental applications. Expression conditions may require optimization depending on the specific construct and bacterial strain used.

How should researchers design dose-response experiments with VEGFA?

When designing dose-response experiments with VEGFA, researchers should consider:

• Concentration range: Evidence suggests testing a wide range (10-200 nM) as different biological effects show distinct dose-response relationships

• Time points: VEGFA responses are time-dependent with threshold and peak activation varying with concentration

• Cell type specificity: Different cell types (endothelial vs. non-endothelial) may have different sensitivity thresholds

• Measurement parameters: Select appropriate readouts based on expected effects (proliferation, migration, gene expression, etc.)

Experimental evidence demonstrates that VEGFA effects on metabolic pathways in hepatocytes follow dose-dependent patterns, with some genes (fas, acc) responsive across concentration ranges (10-200 nM), while others (scd1) only respond at higher concentrations (200 nM) . Similarly, glycolysis-related genes (pk, gk, gys2) show significant upregulation only at higher doses (100-200 nM) .

For gradient studies, microvascular endothelial cells can distinguish as little as 3-fold differences in VEGFA concentration, indicating the need for careful concentration control in experiments .

What experimental approaches can assess VEGFA function in metabolic regulation?

To investigate VEGFA's role in metabolic regulation, researchers can employ several approaches:

• Measurement of lipid and glycogen accumulation in target cells after VEGFA treatment

• Analysis of metabolic gene expression (e.g., fas, acc, scd1 for lipid metabolism; pk, gk, gys2 for glucose metabolism)

• Assessment of pathway activation through phosphorylation status (particularly AMPKα at T172)

• Pathway inhibition studies (e.g., using Compound C to inhibit AMPKα) to validate mechanistic hypotheses

Studies in hepatocytes demonstrate that VEGFA significantly increases lipid and glycogen accumulation in a dose-dependent manner, with effects observable from 10 to 200 μM . This effect appears to be mediated through AMPKα signaling, as inhibition of this pathway with Compound C attenuates VEGFA-induced triglyceride and glycogen accumulation .

How does VEGFA function in liver regeneration models?

VEGFA plays a complex role in liver regeneration that varies depending on the underlying health of the liver. In models of partial hepatectomy with vascular occlusion (PH + I/R):

This suggests that VEGFA modulation strategies should differ based on underlying metabolic conditions. The finding that VEGFA inhibition promotes regeneration specifically in T2DM settings highlights the importance of considering comorbidities in experimental design .

For researchers studying liver regeneration:

• Different intervention points may yield opposite outcomes

• Source tissues (intestine, adipose tissue) influence hepatic VEGFA levels

• Effects on related growth factors (e.g., VEGFB) should be monitored simultaneously

What considerations are important when studying VEGFA in ischemic disease models?

Timing is critical when studying VEGFA in ischemic contexts. Evidence from stroke models demonstrates:

• Administration timing is crucial - VEGFA delivery 48 hours after ischemic stroke increases revascularization and improves neurological outcomes

• Acute administration (immediately after ischemic event) increases blood-brain-barrier leakage and elevates hemorrhagic transformation risk

This demonstrates a narrow therapeutic window where VEGFA modulation can be either beneficial or harmful. Researchers should therefore:

• Include multiple intervention timepoints in study designs

• Monitor both beneficial (revascularization) and adverse (increased permeability) outcomes

• Consider combinatorial approaches that mitigate risks while preserving benefits

How do VEGFA gradient effects influence experimental design?

VEGFA gradients are critical during development and may be important in various experimental contexts. Research indicates that microvascular endothelial cells can perceive and interpret graded VEGFA signals with remarkable precision:

• Threshold and time to peak activation of VEGFR2 depend on signal strength over a 50-fold concentration range

• Cells can distinguish even 3-fold differences in concentration

• Signal intensity and duration are not interpreted equivalently - longer exposure does not compensate for low concentration

These findings have important implications for experimental design:

• When creating VEGFA gradients in vitro or in vivo, ensure concentration differences are precisely controlled

• Consider both spatial and temporal aspects of VEGFA signaling

• Account for the non-equivalence of concentration and duration in interpreting results

• Design appropriate controls that distinguish between gradient effects and absolute concentration effects

What are key considerations for anti-VEGFA therapeutic approaches?

The development of anti-VEGFA therapies has yielded important insights for researchers:

• Anti-VEGFA treatments may have tissue-specific complications:

  • In kidney glomeruli, VEGFA inhibition can lead to thrombotic microangiopathy phenotypes

  • In cancer contexts, anti-VEGFA agents often show initial benefits followed by resistance development

• Combinatorial approaches may enhance efficacy:

  • Combining low doses of anti-VEGFA agents with chemotherapeutics (e.g., vinblastine) led to sustained tumor regression without increased toxicity

  • This suggests VEGFA inhibition may particularly affect endothelial cell recovery/survival following damage

• Therapeutic window considerations:

  • Anti-VEGFA treatments must balance potential benefits against risks

  • Biomarkers predicting response or complications remain an active research area

These observations highlight the importance of careful response monitoring, concentration optimization, and combination strategies when studying VEGFA modulation in disease models.

How can researchers study VEGFA genetic variation in disease contexts?

When investigating VEGFA genetic variants in disease, researchers should:

• Select appropriate polymorphisms to genotype:

  • Common polymorphisms include rs699947 (–1540C>A), rs833061 (–460T>C), rs2010963 (405C>G), rs25648 (1032C>T) and rs3025039 (1689C>T)

  • Minor allele frequencies typically exceed 5% in populations

• Use reliable genotyping methods:

  • TaqMan MGB probes with allelic discrimination via real-time PCR

  • Validate selected samples through DNA sequencing

• Apply appropriate statistical approaches:

  • Analyze linkage disequilibrium using programs like Haploview

  • Estimate haplotypes using Bayesian algorithms (e.g., Phase program)

  • Apply multiple genetic models (additive, dominant, recessive) to detect associations

  • Use appropriate statistical tests (χ² test for distribution differences; Cox regression for survival analyses)

• Consider clinical correlations:

  • Analyze associations with disease features, progression, and outcomes

  • Include relevant clinical variables in multivariate models