VEGF Mouse, Sf9

What is VEGF Mouse, Sf9 and what are its structural characteristics?

VEGF Mouse, Sf9 is a recombinant Vascular Endothelial Growth Factor from mouse produced in Sf9 insect cells using baculovirus expression system. It is a double, glycosylated polypeptide chain containing 164 amino acids with a molecular mass of 48 kDa. The protein is purified through proprietary chromatographic techniques and typically supplied as a sterile filtered white lyophilized powder with greater than 95% purity as determined by RP-HPLC and SDS-PAGE analysis .

The protein corresponds to UniProt entry Q00731 and is also known by several alternative names including Vascular endothelial growth factor A, VEGF-A, Vascular permeability factor (VPF), and MGC70609 .

What is the biological activity of VEGF Mouse, Sf9 and how is it measured?

The biological activity of VEGF Mouse, Sf9 is determined through its ability to stimulate proliferation of human umbilical vein endothelial cells (HUVEC). The ED50 range (effective dose for 50% response) is typically 1-2 ng/ml as measured by 3H-thymidine uptake. This corresponds to a specific activity of 0.5-1 MIU/mg (Million International Units per milligram) .

This standardized bioassay provides a reliable measurement of functional capacity to stimulate endothelial cell proliferation, which is crucial for validating the protein's activity before experimental use. When designing experiments, researchers should consider this activity range when determining appropriate dosing for their specific cellular models.

What are the recommended storage and handling conditions for VEGF Mouse, Sf9?

For optimal stability and activity, the following storage and handling guidelines should be followed:

The lyophilized protein should be reconstituted in sterile 18MΩ-cm H₂O at a concentration of not less than 100μg/ml, which can then be further diluted to other aqueous solutions as needed for specific experimental protocols .

How does VEGF signal through STAT3 to promote vascular permeability, and what experimental models best demonstrate this relationship?

VEGF signaling through Signal Transducer and Activator of Transcription 3 (STAT3) represents a critical pathway for inducing vascular permeability. Mechanistically, VEGF stimulation promotes physical interaction between VEGFR-2 and STAT3, as demonstrated through immunoprecipitation studies showing association between both total and phosphorylated forms of these proteins. This interaction leads to STAT3 phosphorylation and nuclear translocation in endothelial cells .

Two complementary experimental models have effectively demonstrated this relationship:

• Zebrafish model: Heat-inducible VEGF transgenic zebrafish crossed with CRISPR/Cas9-generated STAT3 knockout zebrafish show significantly reduced vascular leakage in the absence of STAT3. The transparency of zebrafish embryos makes them particularly valuable for in vivo fluorescent imaging of VEGF-induced permeability .

• Endothelial-specific STAT3 knockout mice: These mice exhibit markedly decreased VEGF-induced vascular permeability as measured through Evans blue extravasation in a footpad permeability assay. Importantly, STAT3 deficiency reduces permeability without impairing vascular development and function .

These findings suggest targeting STAT3 might offer therapeutic potential for reducing pathological vascular permeability while preserving normal vascular function.

What does computational modeling reveal about VEGF distribution in mouse models and how can this inform experimental design?

Computational modeling of VEGF distribution in mice reveals several important insights that should inform experimental design:

• The concentration of unbound VEGF in tissue is approximately 50-fold greater than in blood, creating distinct microenvironments that may respond differently to interventions .

• The multiscale model accounts for interactions between two major mouse VEGF isoforms (VEGF120 and VEGF164) and their endothelial cell receptors VEGFR-1, VEGFR-2, and co-receptor neuropilin-1, which is also expressed on parenchymal cells .

• VEGF concentration is highly dependent on the secretion rate, which can be estimated through parameter fitting to experimental data - a value difficult to measure directly through experiments .

This computational approach provides quantitative interpretation of animal data and can be used alongside experimental studies in developing pro- and anti-angiogenic agents. The model helps bridge the gap between mouse studies (preclinical) and human studies (clinical trials) by providing a framework for scaling-up therapeutics .

How does VEGF interact with bone formation pathways, and what molecular mechanisms connect angiogenesis with osteogenesis?

The relationship between VEGF and bone formation represents a crucial example of coupled angiogenesis and osteogenesis. Genetic manipulations in mice have provided evidence for a critical role of VEGF in this coupling mechanism .

A key molecular connection is through the transcription factor Osterix (Osx), which is indispensable for osteoblast differentiation. Research demonstrates that Osx regulates VEGF promoter activity in a dose-dependent manner, with increasing amounts of Osx inducing markedly higher VEGF promoter activities. Transfection experiments show that 800 ng of Osx can result in a 20-fold increase in VEGF promoter activity .

In conditional Osx-null mice, immunohistochemistry reveals markedly reduced VEGF protein levels in osteoblasts compared to wild-type controls. The trabecular bone in tibiae of these mutants becomes disorganized with significantly reduced volume .

This molecular relationship highlights how bone development requires coordinated vascular invasion, with VEGF serving as a critical coupling factor between osteoblast activity and angiogenesis.

What are the methodological approaches for studying VEGF-induced vascular permeability in animal models?

Several methodological approaches are available for studying VEGF-induced vascular permeability in animal models:

• Mouse footpad permeability assay:

  • Intravenous injection of Evans blue dye followed by subcutaneous injection of recombinant VEGF (2.5 μg/ml in PBS) in one footpad and vehicle in the contralateral footpad

  • After 30 minutes, footpads are excised, extravasated dye extracted via formamide, and measured by spectrometry

  • This method provides quantitative assessment of VEGF-induced vascular permeability

• Zebrafish fluorescent microangiography:

  • Use of transgenic heat-inducible VEGF zebrafish allows temporal control of VEGF expression

  • Microinjection of fluorescent dextran mixtures (e.g., 2000 kDa FITC-dextran and 70 kDa Texas Red-dextran) into the ventricle

  • Differential extravasation of different sized dextrans indicates permeability changes

  • Transparent embryos allow direct visualization of vascular leakage

• Two-compartment modeling approach:

  • Computational models of VEGF distribution between blood and tissue compartments

  • Accounts for transcapillary macromolecular permeability, lymphatic transport, and plasma clearance

  • Provides quantitative interpretation framework for experimental permeability data

These complementary approaches provide robust assessment of vascular permeability across different experimental systems.

What factors should be considered when designing experiments to study VEGF-receptor interactions?

When designing experiments to study VEGF-receptor interactions, researchers should consider several key factors:

• Receptor expression profiles:

  • VEGF interacts with multiple receptors including VEGFR-1, VEGFR-2, and co-receptor neuropilin-1

  • Receptors may be expressed on both luminal and abluminal surfaces of endothelial cells

  • Neuropilin-1 is also expressed on parenchymal cells (e.g., myocytes)

  • Verify receptor expression in your specific model system

• VEGF isoform selection:

  • Mouse has different major isoforms (VEGF120 and VEGF164) compared to human

  • Different isoforms may have distinct binding properties and biological activities

  • Select appropriate isoform(s) for your research question

• Detection methods:

  • Immunoprecipitation studies can demonstrate physical association between receptors and downstream signaling molecules

  • GST pull-down experiments help characterize protein-protein interactions

  • Immunofluorescence visualizes receptor activation and nuclear translocation of signaling molecules

• Functional readouts:

  • Measure endothelial cell proliferation using 3H-thymidine uptake or other proliferation assays

  • Assess vascular permeability using the methods described in FAQ 3.1

  • Evaluate downstream signaling through phosphorylation status of key pathway components

Careful consideration of these factors ensures robust and reproducible study of VEGF-receptor interactions.

How can researchers optimize reconstitution and dilution protocols for VEGF Mouse, Sf9 to maintain consistent activity?

Optimizing reconstitution and dilution protocols is critical for maintaining consistent VEGF activity:

• Initial reconstitution:

  • Use sterile 18MΩ-cm H₂O for reconstitution

  • Maintain a minimum concentration of 100 μg/ml during reconstitution to ensure protein stability

  • Reconstitute gently by swirling rather than vigorous vortexing to prevent protein denaturation

• Buffer selection for dilution:

  • After initial reconstitution in water, the solution can be further diluted in appropriate buffers

  • Select buffers that maintain physiological pH (7.2-7.4) and ionic strength

  • Consider adding protein carriers for dilute solutions

• Stability enhancers:

  • For long-term storage, add carrier protein (0.1% HSA or BSA) to prevent protein adsorption to tubes and enhance stability

  • Aliquot reconstituted protein to avoid repeated freeze-thaw cycles

• Quality control:

  • Validate activity using standardized bioassays such as HUVEC proliferation

  • Expected ED50 should be within 1-2 ng/ml range

  • Purity can be verified through RP-HPLC and SDS-PAGE analysis

• Storage conditions:

  • Store reconstituted protein at 4°C if using within 2-7 days

  • For longer storage, keep below -18°C with carrier protein addition

  • Always transport on blue ice

Following these optimized protocols ensures consistent biological activity across experiments.

What are common challenges in VEGF-induced angiogenesis assays and how can researchers address them?

Researchers frequently encounter several challenges when conducting VEGF-induced angiogenesis assays:

• Variability in endothelial cell responsiveness:

  • Challenge: Different endothelial cell sources and passage numbers may respond differently to VEGF stimulation

  • Solution: Standardize cell sources, limit passage numbers, and include positive controls in each experiment to normalize responses

• Dose-response inconsistencies:

  • Challenge: The effective concentration range may vary between different experimental setups

  • Solution: Perform comprehensive dose-response studies starting near the expected ED50 (1-2 ng/ml) and extending above and below this range

• Distinguishing direct and indirect effects:

  • Challenge: VEGF may act directly on endothelial cells but also affect other cell types that influence angiogenesis

  • Solution: Use co-culture systems and conditional knockout models to dissect cell-specific responses

• Translating between in vitro and in vivo findings:

  • Challenge: In vitro responses may not accurately predict in vivo outcomes due to complex microenvironmental factors

  • Solution: Utilize complementary approaches and validate key findings across multiple model systems, considering the 50-fold higher VEGF concentration in tissue versus blood

• Background angiogenesis:

  • Challenge: Baseline angiogenic activity may mask VEGF-specific effects

  • Solution: Include appropriate vehicle controls and consider using angiogenesis-deficient backgrounds for clearer signal detection

Addressing these challenges through careful experimental design improves reproducibility and interpretability of VEGF-induced angiogenesis assays.

How should researchers interpret differences in VEGF activity between species and cellular contexts?

When interpreting differences in VEGF activity across species and cellular contexts, researchers should consider several important factors:

• Species-specific isoform differences:

  • Mouse expresses VEGF120 and VEGF164 as major isoforms versus human VEGF121 and VEGF165

  • These differences can affect receptor binding affinity and extracellular matrix interactions

  • When translating between mouse and human systems, account for these molecular distinctions

• Receptor expression patterns:

  • Receptor distribution varies between tissue types and species

  • Consider the balance between different receptors (VEGFR-1, VEGFR-2, neuropilin-1)

  • Neuropilin-1 expression on both endothelial cells and parenchymal cells adds complexity to interpretation

• Tissue-specific microenvironments:

  • The 50-fold concentration difference between tissue and blood compartments creates distinct signaling contexts

  • Matrix components, hypoxia, and co-expressed factors modulate VEGF activity

  • Consider tissue-specific factors when comparing across experimental systems

• Developmental versus adult systems:

  • VEGF may have different roles during development compared to adult physiology

  • Some animal models demonstrate that STAT3 deficiency reduces VEGF-induced permeability without impairing vascular development, highlighting context-dependent functions

Using computational models that account for these differences can help bridge experimental findings across species and contexts, particularly when scaling from preclinical to clinical applications .

What are the key considerations for validating novel findings about VEGF signaling mechanisms?

Validating novel findings about VEGF signaling mechanisms requires a multi-faceted approach:

• Complementary experimental systems:

  • Validate key findings across multiple models (cell lines, primary cells, animal models)

  • Zebrafish and mouse models offer complementary strengths - zebrafish for visualization, mice for mammalian relevance

  • Consider both in vitro and in vivo validation approaches

• Genetic manipulation strategies:

  • Use both gain-of-function and loss-of-function approaches

  • Compare acute knockdown versus genetic knockout effects

  • Consider conditional and tissue-specific manipulation to avoid developmental effects

  • CRISPR/Cas9 genomic knockout zebrafish and endothelial-specific knockout mice provide complementary genetic approaches

• Molecular mechanism confirmation:

  • Demonstrate physical interactions between signaling components (e.g., VEGFR-2 and STAT3)

  • Validate using multiple techniques (immunoprecipitation, GST pull-down, immunofluorescence)

  • Confirm subcellular localization changes (e.g., nuclear translocation of transcription factors)

• Functional validation:

  • Connect molecular events to functional outcomes (permeability, proliferation, etc.)

  • Quantify effects using standardized assays (Evans blue extravasation, 3H-thymidine uptake)

  • Demonstrate dose-dependency and specificity through appropriate controls

• Computational modeling:

  • Use mathematical models to integrate experimental data

  • Test whether proposed mechanisms quantitatively explain observed behaviors

  • Identify key parameters through sensitivity analysis

Thorough validation across these dimensions strengthens novel findings about VEGF signaling mechanisms and increases their potential translational impact.

How can VEGF Mouse, Sf9 be utilized to study the relationship between angiogenesis and osteogenesis?

VEGF Mouse, Sf9 offers valuable opportunities to study the angiogenesis-osteogenesis relationship:

• Molecular regulatory mechanisms:

  • Investigate how transcription factors like Osterix regulate VEGF expression in osteoblasts

  • Perform promoter activity assays to quantify dose-dependent effects on VEGF transcription

  • Explore how VEGF subsequently influences both vascular and skeletal cell behaviors

• Conditional knockout models:

  • Utilize conditional Osx knockout mice to examine VEGF expression in mature osteoblasts

  • Compare immunohistochemical VEGF staining patterns in tibiae of control versus knockout mice

  • Correlate VEGF levels with trabecular bone organization and volume

• Co-culture systems:

  • Develop co-culture models of osteoblasts with endothelial cells

  • Add recombinant VEGF Mouse, Sf9 at defined concentrations to study dose-dependent effects

  • Measure both angiogenic (endothelial proliferation, migration, tube formation) and osteogenic (mineralization, bone marker expression) outcomes

• In vivo models:

  • Create local VEGF gradients using controlled release systems in bone defect models

  • Use transgenic models with VEGF overexpression or conditional deletion in osteoblasts

  • Employ fluorescent reporter systems to simultaneously visualize vascular invasion and bone formation

These approaches will help elucidate the molecular coupling between angiogenesis and osteogenesis, with implications for bone tissue engineering and regenerative medicine.

What are the emerging techniques for studying VEGF distribution and activity in three-dimensional tissue models?

Several innovative techniques are emerging for studying VEGF in three-dimensional contexts:

• Microfluidic organ-on-chip platforms:

  • Create perfusable vascular networks within 3D extracellular matrices

  • Introduce controlled VEGF gradients to study directional angiogenic responses

  • Monitor real-time changes in barrier function and vascular permeability

  • Recapitulate the complex tissue-blood distribution patterns revealed by computational modeling

• Advanced imaging approaches:

  • Utilize two-photon microscopy for deeper tissue penetration in 3D models

  • Apply light sheet microscopy for rapid volumetric imaging with minimal phototoxicity

  • Implement intravital microscopy in zebrafish models to visualize VEGF-induced permeability in real-time

• Biomaterial-based delivery systems:

  • Develop hydrogels with controlled VEGF release profiles

  • Create spatially patterned VEGF gradients to direct vascular growth

  • Design materials with cell-triggered VEGF release mechanisms

• Biosensor technology:

  • Generate FRET-based biosensors to visualize VEGF receptor activation

  • Develop reporter systems for downstream signaling events like STAT3 activation

  • Create tension sensors to monitor VEGF-induced changes in endothelial junctions

These techniques will provide more physiologically relevant insights into VEGF biology than traditional 2D culture systems and help bridge the gap between in vitro and in vivo findings.

How might computational modeling be used to predict therapeutic responses to VEGF-targeted interventions?

Computational modeling offers powerful approaches for predicting therapeutic responses to VEGF-targeted interventions:

• Multiscale integration:

  • Develop models spanning from molecular interactions to tissue-level responses

  • Incorporate the two-compartment (blood and tissue) framework to account for differential VEGF distribution

  • Model interactions between VEGF isoforms and their receptors (VEGFR-1, VEGFR-2, neuropilin-1)

• Parameter estimation and validation:

  • Estimate difficult-to-measure parameters like endogenous VEGF secretion rates

  • Validate model predictions against experimental measurements

  • Perform sensitivity analysis to identify key parameters governing system behavior

• Therapeutic prediction applications:

  • Simulate effects of VEGF-binding drugs with different pharmacokinetic properties

  • Model receptor-specific targeting strategies (VEGFR-1 vs. VEGFR-2)

  • Predict differential effects across tissues based on receptor expression patterns

  • Explore combination therapy approaches targeting multiple nodes in VEGF signaling

• Translation between species:

  • Scale parameters between mouse models and human patients

  • Use mouse models to calibrate and validate computational predictions

  • Apply validated models to predict human clinical responses based on preclinical data