VEGF (121 a.a.) Mouse

What is the molecular structure and basic characteristics of mouse VEGF121?

Mouse VEGF121 is a truncated version of VEGF165, functioning as a homodimeric, non-glycosylated polypeptide chain with a molecular mass of 28.4 kDa. It is one of three major VEGF isoforms found in mice (120, 164, and 188 amino acids). Unlike larger isoforms, VEGF121 lacks the basic heparin-binding regions encoded by exon 7, making it freely diffusible rather than matrix-bound. This diffusibility is due to the absence of 15 basic amino acids within the 44 residues normally encoded by exon 7 . The protein is weakly acidic and demonstrates high sequence conservation across species, sharing 98% identity with rat VEGF121, 89% with canine, feline, equine and porcine variants, and 87% with human, ovine and bovine VEGF .

How does the tissue distribution of VEGF121 in mice compare to other isoforms?

VEGF121 demonstrates distinct tissue-specific expression patterns in mice under normal physiological conditions. It is present in large quantities in the kidneys and lungs, while only low levels are observed in the heart and brain . Recent studies have also found abundant levels of VEGF121 compared to other VEGF isoforms in the rabbit anterior cruciate ligament . This differential expression pattern suggests tissue-specific functions for this particular isoform. In pathological states, especially in cancer models, the relative balance of isoform expression often shifts toward increased VEGF121 production, as demonstrated in prostate and colon cancer tissues .

What are the receptor binding properties of mouse VEGF121?

Mouse VEGF121 shows preferential binding to the larger VEGF receptors, VEGFR-1 and VEGFR-2, though with lower receptor-binding affinity than VEGF165 . Its interaction with neuropilins (Np-1 and Np-2) is more complex than initially thought. While early research suggested VEGF121 did not specifically interact with Np-1, more recent studies have demonstrated that neuropilins can enhance VEGF121-stimulated signal transduction via the phosphorylation of VEGFR-2 . Further research has shown that blocking functional Np-1 receptors reduced VEGF121-induced endothelial cell migration and sprout formation, indicating a functional relationship . Though VEGF121 can bind directly to Np-1, this interaction alone appears insufficient to create the Np-1/VEGFR-2 complex that mediates some signaling outcomes .

How does mouse VEGF121 concentration differ between blood and tissue compartments?

Computational modeling of VEGF distribution in mice indicates significantly higher concentrations of unbound VEGF (including VEGF121) in tissue compared to blood circulation. Simulation studies predict that the concentration of unbound VEGF in tissue is approximately 50-fold greater than in blood . These concentration gradients are highly dependent on the VEGF secretion rate, which varies by tissue type and physiological state. The significant difference between tissue and blood concentrations has important implications for experimental design when studying VEGF121 functions, as sampling from different compartments will yield substantially different measurements .

What methodological approaches should be used to study VEGF121's role in angiogenesis compared to other isoforms?

When investigating VEGF121's specific role in angiogenesis, researchers should employ comparative methodologies that account for its unique diffusibility characteristics. The chorioallantoic membrane (CAM) assay represents an effective model system for such comparisons. Studies have demonstrated that the spatial distribution of VEGF isoforms significantly impacts vessel morphology and network organization . When designing experiments, researchers should:

• Compare matrix-bound versus soluble delivery systems to account for VEGF121's freely diffusible nature

• Implement quantitative vessel analysis examining multiple parameters including:

  • Arterial and venous branching patterns

  • Capillary plexus organization

  • Endothelial cell morphology

  • Periendothelial cell interactions

  • Vessel permeability characteristics

Research has shown that cell-demanded release of engineered VEGF121 from matrices (e.g., fibrin implants) induces more organized vascular growth with normal morphologies at both light microscopic and ultrastructural levels compared to passive diffusion of native VEGF121, which primarily induces chaotic changes within the capillary plexus .

How can recombinant VEGF121 be effectively used in mouse models of preeclampsia?

VEGF121 has shown therapeutic potential in mouse models of preeclampsia, particularly those induced by AT1 receptor autoantibodies (AT1-AA). For effective experimental implementation, consider the following methodology:

• Delivery method: Continuous infusion via osmotic minipump implanted subcutaneously has proven effective in maintaining stable VEGF121 levels .

• Dosing parameters: Effective doses should be determined based on the specific model and outcome measurements.

• Key outcome measurements:

  • Blood pressure (can decrease from 159 ± 5 to 124 ± 5 mm Hg with VEGF121 treatment)

  • Proteinuria

  • Renal function (BUN levels)

  • Kidney histopathology

  • Placental damage assessment

  • Endothelial cell content in placenta

VEGF121 infusion has been demonstrated to attenuate AT1-AA-induced hypertension and proteinuria in pregnant mice, as well as reduce kidney damage, renal dysfunction, and placental impairment . The therapeutic benefit appears to be mediated through neutralization of excess soluble Flt-1 (sFlt-1), which is elevated in preeclampsia models .

What are the critical considerations when designing experiments to study VEGF121 signaling mechanisms?

When investigating VEGF121 signaling mechanisms, researchers should consider several critical factors:

• Receptor interactions: Design experiments that can differentiate between direct VEGFR-1/VEGFR-2 activation and neuropilin-mediated enhancement of signaling. This may require:

  • Receptor-specific blocking antibodies

  • Genetic knockdown/knockout of specific receptors

  • Phosphorylation analysis of different receptor types

• Downstream pathway analysis: VEGF121 activates multiple signaling cascades that may differ from other isoforms. Consider examining:

  • MAPK pathway activation

  • PI3K/Akt signaling

  • eNOS phosphorylation

  • Calcium signaling

• Diffusion characteristics: Account for VEGF121's high diffusibility by:

  • Using appropriate matrices or delivery systems

  • Comparing gradient-forming versus uniform distribution conditions

  • Implementing VEGF121 variants with modified diffusibility properties

• Temporal dynamics: VEGF121 signaling may show different kinetics compared to matrix-bound isoforms, requiring time-course studies with appropriate sampling intervals .

How do computational models inform experimental design for studying VEGF121 distribution?

Computational modeling provides valuable insights for experimental design when studying VEGF121 distribution and activity. Two-compartment models of VEGF distribution in mice suggest:

• Parameter sensitivity considerations:

  • VEGF secretion rate is a critical determinant of tissue and blood concentrations

  • Transcapillary macromolecular permeability affects distribution between compartments

  • Receptor densities on different cell types influence local VEGF availability

• Sampling strategy implications:

  • Due to the ~50-fold higher tissue versus blood concentration, tissue sampling provides better detection sensitivity

  • Blood sampling requires more sensitive assays but may better reflect systemic effects

• Experimental validation approaches:

  • Parallel measurements from multiple compartments help validate model predictions

  • Perturbation experiments (e.g., receptor blockade) can test model predictions about system responses

  • Tagged VEGF121 variants can track distribution patterns predicted by the model

Such models can provide quantitative interpretation of preclinical data and assist in developing pro- and anti-angiogenic agents by predicting distribution dynamics that might be difficult to measure experimentally .

What methodologies are most effective for studying the differential effects of VEGF121 versus other isoforms on vessel morphology?

To effectively study how VEGF121 influences vessel morphology differently from other isoforms, researchers should implement multiscale analysis techniques:

• Macro-level analysis:

  • Quantitative assessment of vessel density, branching patterns, and network complexity

  • Arterial versus venous development quantification

  • In vivo imaging of vascular networks (e.g., using intravital microscopy)

• Cellular-level analysis:

  • Endothelial cell morphology and alignment

  • Pericyte recruitment and coverage

  • Endothelial junction formation and integrity

  • Cell proliferation versus migration contributions to angiogenesis

• Delivery system considerations:

  • Compare matrix-bound systems (fibrin, collagen, synthetic hydrogels) versus soluble delivery

  • Engineered VEGF121 variants with controlled release properties

  • Gradient-forming versus uniform distribution systems

Research has demonstrated that cell-demanded release of engineered VEGF121 (e.g., α2PI1–8-VEGF121) from fibrin matrices promotes more organized vessel formation with normal morphology compared to passively released wild-type VEGF121, which primarily induces disorganized changes in the capillary plexus .

How can VEGF121 be effectively used in therapeutic angiogenesis research?

For therapeutic angiogenesis research using VEGF121, consider the following methodological approaches:

• Delivery optimization:

  • Engineer matrix-binding variants (e.g., α2PI1–8-VEGF121) for controlled release

  • Utilize biomaterial systems that protect VEGF121 from clearance

  • Implement cell-demanded release mechanisms through incorporation of protease-sensitive linkers

• Outcome assessment parameters:

  • Vessel functionality (perfusion studies)

  • Vessel maturation and stability (pericyte coverage)

  • Vessel permeability (contrast-enhanced imaging or dye extravasation studies)

  • Tissue oxygenation and metabolic effects

  • Long-term stability of induced vasculature

• Disease-specific considerations:

  • For preeclampsia models: focus on placental vascularization and maternal blood pressure

  • For ischemic conditions: tissue perfusion and functional recovery

  • For wound healing: granulation tissue formation and wound closure rates

Studies have shown that vessels induced by engineered VEGF121 with controlled release properties demonstrate reduced leakage and more normal morphology than those induced by native VEGF121, suggesting improved therapeutic potential .

How does VEGF121 expression change in mouse models of cancer, and what are the methodological approaches to study this?

VEGF121 expression undergoes significant alterations in mouse models of cancer, with methodological implications for research:

• Expression analysis techniques:

  • Quantitative real-time PCR for isoform-specific expression profiling

  • In situ hybridization for spatial localization of expression

  • Isoform-specific antibodies for protein detection when available

  • Laser capture microdissection to analyze expression in specific tumor regions

• Documented expression changes:

  • In prostate cancer models, a significant shift toward increased VEGF121 expression compared to VEGF165 occurs during malignant transformation

  • Experimental manipulation to increase relative VEGF121 expression (using antisense technology) can dramatically increase tumor angiogenesis

  • Similar upregulation patterns have been observed in colon cancer models

• Functional analysis approaches:

  • Isoform-specific knockdown studies

  • Introduction of recombinant isoforms

  • Analysis of tumor vascular characteristics in relation to isoform expression profiles

  • Correlation of isoform expression with metastatic potential

Understanding these expression changes and implementing appropriate methodologies is critical for studying VEGF121's specific contribution to tumor angiogenesis and developing targeted therapies .

What are the technical challenges in measuring VEGF121 in mouse models of preeclampsia?

Measuring VEGF121 in mouse models of preeclampsia presents several technical challenges requiring specific methodological approaches:

• Sample collection considerations:

  • Plasma versus tissue measurements yield significantly different concentrations

  • Timing of collection is critical as VEGF121 levels fluctuate throughout pregnancy

  • Local placental concentrations may differ from systemic measurements

• Detection method limitations:

  • Many commercial ELISA kits do not differentiate between VEGF isoforms

  • Cross-reactivity with other VEGF family members must be controlled

  • Free versus receptor-bound VEGF121 may require different extraction methods

• Confounding factors in preeclampsia models:

  • Elevated sFlt-1 binds VEGF, potentially masking detection

  • Changes in blood volume and hemoconcentration affect concentration measurements

  • Altered renal clearance in preeclampsia may impact VEGF121 pharmacokinetics

• Recommended approaches:

  • Use isoform-specific detection methods when possible

  • Include measurements of free and bound VEGF121

  • Compare tissue and plasma levels for comprehensive assessment

  • Account for sFlt-1 levels when interpreting VEGF121 measurements

What are the optimal conditions for storage and handling of recombinant mouse VEGF121 for research applications?

For maintaining optimal activity of recombinant mouse VEGF121 in research applications, adhere to these technical guidelines:

• Storage conditions:

  • Store lyophilized protein at -20°C to -80°C

  • For reconstituted protein, store at -80°C in single-use aliquots

  • Avoid repeated freeze-thaw cycles which significantly decrease bioactivity

  • Protect from light exposure during storage

• Reconstitution parameters:

  • Use sterile buffers appropriate for cell culture applications

  • For maximum stability, reconstitute in solutions containing carrier protein (e.g., 0.1% BSA)

  • Allow complete solubilization before use (gentle swirling rather than vortexing)

  • Filter sterilize if needed for cell culture applications

• Working concentration considerations:

  • Effective concentrations vary by application:

    • Cell culture: 5-100 ng/mL depending on cell type and assay

    • In vivo studies: typically 20-100 μg/kg for systemic administration

    • Local delivery: 10-100 ng per implant site depending on release system

• Compatibility considerations:

  • VEGF121 activity may be affected by binding to certain surfaces

  • Use low-protein binding tubes for storage and handling

  • Consider potential interactions with delivery matrices for in vivo applications

What methods can be used to quantify the bioactivity of mouse VEGF121 preparations?

To ensure research-grade mouse VEGF121 maintains appropriate bioactivity, several quantification methods should be considered:

• Endothelial cell-based assays:

  • Proliferation assays using mouse endothelial cells (3-5 day assay)

  • Migration assays (Boyden chamber or scratch wound healing)

  • Tube formation assays on Matrigel or other matrices (4-24 hour assay)

  • Receptor phosphorylation assays (acute response: 5-30 minutes)

• Molecular binding assays:

  • VEGFR-1 and VEGFR-2 binding ELISAs or surface plasmon resonance

  • Competitive binding assays against standard VEGF preparations

  • Neuropilin binding assessment for functional interactions

• In vivo activity assessment:

  • Chorioallantoic membrane (CAM) assays

  • Matrigel plug assays in mice

  • Mouse corneal micropocket assays

  • Comparison to reference standards for vessel induction potential

• Quality control parameters:

  • Purity assessment by SDS-PAGE (>95% purity recommended)

  • Endotoxin testing (<1 EU/μg protein for in vivo applications)

  • Confirmation of protein concentration by validated methods

  • Stability testing under intended storage conditions

Comparing results across multiple assay systems provides the most comprehensive assessment of bioactivity, with endothelial cell proliferation serving as the primary functional readout for most research applications.