VEGF (121 a.a.) Human, Sf9

What is VEGF (121 a.a.) Human, Sf9 and how does it differ from other VEGF isoforms?

VEGF (121 a.a.) Human, Sf9 is a recombinant form of human Vascular Endothelial Growth Factor A (VEGF-A) containing 121 amino acids, produced in Sf9 insect cells. It exists as an 18kDa homodimer that glycosylates to approximately 36kDa . VEGF-121 differs from other VEGF isoforms (such as VEGF-165, VEGF-189, or VEGF-206) primarily in its binding properties. Unlike larger isoforms, VEGF-121 lacks heparin-binding domains, making it more soluble and allowing it to circulate more freely in tissues rather than binding to extracellular matrix components . This non-heparan sulfate-binding characteristic gives VEGF-121 distinct diffusion properties that affect its biological activity and spatial distribution in tissues.

What receptors does VEGF-121 bind to and with what affinity?

VEGF-121 binds primarily to two receptor tyrosine kinases: KDR (kinase domain receptor)/Flk-1 in mice and FLT-1 (fms-like tyrosine kinase-1) . Studies using ELISA and competitive binding assays have demonstrated that VEGF-121 binds to Flk-1 with high affinity, comparable to native human VEGF-121 . The binding of VEGF-121 to KDR/Flk-1 is particularly significant as it triggers receptor phosphorylation in both early (1-10 minutes) and late (4-8 hours) phases, leading to downstream signaling events . While VEGF-121 can also bind to FLT-1, this interaction appears less significant for mediating cytotoxicity in targeted therapeutic applications, as cells expressing FLT-1 but low levels of KDR show resistance to VEGF-121-based fusion toxins .

How does VEGF-121 induce signal transduction in endothelial cells?

VEGF-121 initiates signaling cascades through binding to its receptors, particularly KDR/Flk-1, triggering receptor dimerization and autophosphorylation. Research has shown that VEGF stimulation promotes physical interaction between VEGFR-2 and STAT3 (Signal Transducer and Activator of Transcription 3), as demonstrated by immunoprecipitation studies and GST pull-down experiments . Upon VEGF binding, STAT3 becomes phosphorylated and translocates to the nucleus, where it regulates gene expression . This VEGF/VEGFR-2/STAT3 signaling axis plays a critical role in vascular permeability, as genetic STAT3 ablation in mouse and zebrafish models has been shown to reduce VEGF-induced vascular permeability without impairing vascular development .

How does receptor density affect VEGF-121 biological activity in endothelial cells?

Research has revealed a critical threshold of KDR/Flk-1 receptor density required for VEGF-121-mediated effects. Cell lines expressing high levels of KDR/Flk-1 (≥1 × 10^5 receptors per cell) demonstrate significantly heightened sensitivity to VEGF-121-based constructs compared to cells with lower receptor expression . The cytotoxicity studies with VEGF-121/rGel fusion proteins revealed striking differences in IC50 values based on receptor density:

This receptor density threshold has important implications for targeting strategies in both experimental and therapeutic contexts . Normal vasculature typically expresses lower levels of KDR/Flk-1 than tumor vasculature, potentially providing a therapeutic window for VEGF-121-targeted approaches.

What is the relationship between VEGF-121 and STAT3 in regulating vascular permeability?

Recent research has established that VEGF-121 signals through STAT3a to promote vascular permeability. Using both genetic STAT3 ablation in mouse models and CRISPR/Cas9-generated STAT3 knockout zebrafish crossed with VEGF-inducible zebrafish, researchers have demonstrated that STAT3 deficiency significantly reduces vascular permeability without compromising normal vascular development and function . Mechanistically, VEGF stimulation promotes physical interaction between VEGFR-2 and STAT3, with both total and phosphorylated forms of these proteins associating in a VEGF-dependent manner . This interaction leads to STAT3 nuclear translocation and subsequent gene expression changes that regulate endothelial barrier function. The identification of this pathway provides potential therapeutic targets for conditions characterized by excessive vascular permeability, such as ischemic stroke, cardiovascular disease, retinal conditions, and COVID-19-associated pulmonary edema .

How can VEGF-121 be effectively used in targeted fusion constructs for research and potential therapeutic applications?

VEGF-121 has proven effective as a targeting moiety in fusion proteins due to its ability to specifically bind to KDR/Flk-1 receptors overexpressed on tumor vasculature. A prominent example is the VEGF-121/rGel fusion construct, which combines VEGF-121 with the toxin gelonin . This construct maintains both the receptor-binding capabilities of VEGF-121 and the translational inhibition activity of gelonin. When designing similar fusion constructs, researchers should consider:

• Linker regions: The VEGF-121/rGel construct used a flexible G4S tether between the components, allowing each portion to maintain its functionality .

• Expression systems: The construct can be expressed as a soluble protein in bacterial systems, though proper folding and dimerization must be verified .

• Targeting selectivity: The fusion protein demonstrates significantly higher cytotoxicity toward cells expressing high levels of KDR/Flk-1 compared to cells with low expression levels or those predominantly expressing FLT-1 .

• In vivo efficacy: Testing in xenograft models showed that the VEGF-121/rGel construct reduced tumor volume to 16% of untreated controls and localized selectively to tumor vessels, causing thrombotic damage with minimal effects on normal vasculature .

What are the optimal storage and handling conditions for VEGF (121 a.a.) Human, Sf9?

For maximum stability and activity of VEGF (121 a.a.) Human, Sf9, adhere to the following protocols:

• Storage of lyophilized protein: While the lyophilized form remains stable at room temperature for up to 3 weeks, long-term storage should be at temperatures below -18°C in a desiccated environment .

• Reconstitution procedure: The lyophilized protein should be reconstituted in PBS or a medium containing at least 0.1% HSA (human serum albumin) or BSA (bovine serum albumin) to achieve a concentration not lower than 50µg/ml . This protein carrier helps maintain stability and prevent adsorption to container surfaces.

• Storage after reconstitution: Reconstituted VEGF-121 can be stored at 4°C for 2-7 days. For longer storage periods, keep below -18°C with the addition of a carrier protein (0.1% HSA or BSA) .

• Avoiding freeze-thaw cycles: Repeated freeze-thaw cycles significantly compromise protein activity and should be strictly avoided . Consider preparing single-use aliquots upon reconstitution.

How can the activity of VEGF-121 be confirmed in research applications?

Several methodologies can verify the biological activity of VEGF-121:

• Proliferation assays: The specific activity of VEGF-121 can be determined through dose-dependent stimulation of human umbilical vein endothelial cell (HUVEC) proliferation. A concentration range of 2-5ng/ml typically corresponds to a specific activity of 200,000-500,000 IU/mg .

• Receptor phosphorylation assays: Functional VEGF-121 induces phosphorylation of KDR/Flk-1 receptors in a biphasic manner, with detectable phosphorylation occurring at both early (1-10 minutes) and late (4-8 hours) time points after exposure .

• Binding assays: ELISA-based binding assays using immobilized receptors can confirm the binding capability of VEGF-121. Competition with unlabeled VEGF-121 can verify binding specificity .

• Purity assessment: The purity of VEGF-121 preparations should be >90% as determined by RP-HPLC and SDS-PAGE analysis .

What controls should be implemented in VEGF-121 experiments to ensure valid results?

To ensure experimental rigor when working with VEGF-121, implement these essential controls:

• Receptor expression validation: Before conducting experiments evaluating VEGF-121 effects, quantify the expression levels of KDR/Flk-1 and FLT-1 receptors on target cells. This characterization is crucial as cellular responses to VEGF-121 are highly dependent on receptor density, with a threshold of approximately 1 × 10^5 receptors per cell required for significant effects .

• Cell state controls: Account for the proliferative state of endothelial cells. Studies have shown that dividing endothelial cells overexpressing KDR are approximately 60-fold more sensitive to VEGF-121-based constructs than non-dividing cells .

• Specificity controls: Include competitive binding experiments using excess unlabeled VEGF-121 to confirm that observed effects are receptor-mediated rather than non-specific .

• Negative controls: Incorporate cell lines lacking VEGF receptors to differentiate between receptor-dependent and receptor-independent effects .

• Positive controls: Include cells known to be responsive to VEGF-121 (such as PAE/KDR cells in log phase) to validate the activity of your protein preparation .

How can zebrafish models be utilized for studying VEGF-121 function in vascular development and permeability?

Zebrafish (Danio rerio) offer exceptional advantages for studying VEGF-121 function due to their genetic similarity to humans and embryonic transparency that enables real-time visualization of vascular processes. A sophisticated experimental approach involves:

• Generating transgenic models: Utilize heat-inducible VEGF transgenic zebrafish crossed with CRISPR/Cas9-generated STAT3 genomic knockout zebrafish to study the role of VEGF-STAT3 signaling in vascular permeability .

• Microinjection techniques: At the 1-cell stage, microinject embryos with Cre mRNA (12.5 ng/μl) to enable genetic modifications in transgenic lines .

• Transgene identification: Identify zebrafish expressing the VEGF-inducible transgene through fluorescent imaging, looking for eGFP expression in the eyes .

• Heat shock induction: At 2 days post-fertilization (dpf), perform a 37°C heat shock of eGFP+ eyed zebrafish to confirm VEGF transgene activity via the absence of mCherry fluorescence .

• Permeability assessment: At 3 dpf, conduct fluorescent microangiography by inserting a microneedle through the pericardium into the ventricle and injecting a mixture of different molecular weight dextrans (2000 kDa FITC-dextran and 70 kDa Texas Red-dextran at 2 mg/ml) .

• VEGF induction: Immediately prior to imaging, perform a 10-minute 37°C heat shock to induce the VEGF transgene .

This experimental design allows researchers to visualize and quantify vascular permeability changes in response to VEGF-121 induction while assessing the impact of genetic modifications to signaling pathways.

What approaches can be used to study the interaction between VEGF-121 and its receptors?

Multiple complementary techniques can elucidate the interactions between VEGF-121 and its receptors:

• Immunoprecipitation (IP): Use anti-VEGFR-2 or anti-STAT3 antibodies to pull down protein complexes after VEGF-121 stimulation, followed by immunoblotting to detect interaction partners. This approach has successfully demonstrated the physical association between total VEGFR-2 and total STAT3, as well as between phosphorylated forms of these proteins .

• GST pull-down assays: Employ GST-tagged STAT3 protein as "bait" and VEGF-121-stimulated endothelial cell lysates as a source of "prey" proteins. This technique has confirmed growth factor-dependent interactions between endothelial cell-derived VEGFR-2 and GST-tagged STAT3 .

• ELISA-based binding assays: Immobilize receptors (e.g., Flk-1) on ELISA plates and assess binding of VEGF-121 or VEGF-121 fusion constructs. Competition with unlabeled VEGF-121 can verify binding specificity .

• Receptor phosphorylation analysis: Monitor the phosphorylation status of receptors following VEGF-121 exposure using phospho-specific antibodies in Western blotting. This approach has revealed biphasic phosphorylation patterns with early (1-10 minutes) and late (4-8 hours) phases .

• Immunofluorescence microscopy: Track the cellular localization of receptors and downstream signaling molecules (such as STAT3 nuclear translocation) following VEGF-121 stimulation .

How should in vitro cytotoxicity assays be designed to evaluate VEGF-121-based targeted therapeutics?

When designing cytotoxicity assays for VEGF-121-based therapeutics (such as VEGF-121/rGel), consider the following methodological aspects:

• Cell line selection: Include multiple endothelial cell lines with varying levels of KDR/Flk-1 and FLT-1 expression. Based on previous research, cells expressing >1 × 10^5 KDR/Flk-1 receptors per cell (e.g., PAE/KDR, bEnd.3) show high sensitivity, while those with lower receptor levels or predominantly expressing FLT-1 (e.g., HUVEC under normoxia, PAE/FLT-1) exhibit resistance .

• Growth conditions: Evaluate cells in both logarithmic growth phase and confluent states, as receptor sensitivity differs significantly (e.g., log-phase PAE/KDR cells show an IC50 of 0.5nM compared to 30nM for confluent cells) .

• Dose-response curves: Test a wide concentration range to accurately determine IC50 values, typically spanning at least 0.001-1000nM .

• Control treatments: Include the toxin component alone (e.g., rGel) to calculate the targeting index (IC50 ratio of toxin alone to fusion construct), which quantifies the targeting advantage provided by the VEGF-121 component .

• Duration: Conduct assays over sufficient time periods (typically 72 hours) to observe the full cytotoxic effect .

• Readout methods: Employ at least two independent viability assessment methods (e.g., MTT assay, trypan blue exclusion, or ATP quantification) to confirm results.

How should researchers interpret differences in VEGF-121 sensitivity across cell types?

When analyzing differential responses to VEGF-121 across cell types, consider these interpretive frameworks:

• Receptor density analysis: The most significant factor determining cellular sensitivity to VEGF-121 is the density of KDR/Flk-1 receptors. Establish a quantitative relationship between receptor numbers and response magnitude. Cells expressing >1 × 10^5 KDR/Flk-1 receptors per cell demonstrate significantly higher sensitivity to VEGF-121-based constructs .

• Receptor type dominance: Distinguish between effects mediated by different receptor types. Evidence indicates that KDR/Flk-1, rather than FLT-1, primarily mediates cytotoxicity of VEGF-121-based fusion toxins, despite both being capable of binding VEGF .

• Cellular context interpretation: Proliferative status significantly influences VEGF-121 sensitivity. Data shows that dividing endothelial cells overexpressing KDR are approximately 60-fold more sensitive to VEGF-121/rGel than non-dividing cells . This proliferation-dependent sensitivity has important implications for targeting tumor vasculature versus normal quiescent endothelium.

• Targeting index calculation: Calculate and compare the targeting index (ratio of IC50 values of toxin alone versus VEGF-121-conjugated toxin) across cell types to quantify the targeting advantage provided by VEGF-121. Higher indices indicate greater targeting specificity .

What are the limitations when extrapolating in vitro VEGF-121 findings to in vivo contexts?

Several critical considerations must guide the translation of in vitro VEGF-121 findings to in vivo situations:

• Pharmacokinetic differences: In vitro systems cannot replicate the complex pharmacokinetics affecting VEGF-121 in vivo, including distribution, metabolism, and clearance. The relatively small size of VEGF-121 (36kDa) and its freely circulating nature due to lack of heparan sulfate binding may result in different tissue penetration and retention compared to larger VEGF isoforms .

• Receptor expression heterogeneity: Receptor expression in vivo is heterogeneous across tissues and can be altered by pathological conditions. While in vitro studies may characterize effects on cells with defined receptor levels, in vivo contexts present mixed populations with varying receptor expression .

• Microenvironmental factors: The tumor microenvironment contains multiple cell types, extracellular matrix components, and soluble factors that may modulate VEGF-121 effects. For instance, hypoxia in tumors can upregulate VEGF receptors, potentially enhancing sensitivity to VEGF-121-based therapeutics beyond what in vitro models predict .

• Compensatory mechanisms: In vivo systems may exhibit compensatory responses to VEGF-121 intervention, including upregulation of alternative angiogenic pathways or receptor expression changes, which are difficult to model in vitro .

• Technical validation: When moving from in vitro to in vivo studies, ensure that fluorescent microangiography or other visualization techniques are appropriately calibrated to detect the range of permeability changes expected based on in vitro data .

How can conflicting data on VEGF-121 signaling mechanisms be reconciled in research?

When faced with conflicting data regarding VEGF-121 signaling mechanisms, implement these analytical strategies:

• Receptor threshold effects: Apparent contradictions in VEGF-121 responses may be explained by the threshold effect of receptor density. Results that seem conflicting could reflect different receptor expression levels rather than true mechanistic differences .

• Temporal signaling dynamics: VEGF-121 induces biphasic receptor phosphorylation (early: 1-10 minutes; late: 4-8 hours), so conflicting observations may result from different sampling timepoints. Ensure comprehensive time-course studies to capture the complete signaling profile .

• Isoform-specific effects: Compare conflicting results against data from other VEGF isoforms to determine if discrepancies are isoform-specific. VEGF-121's unique lack of heparin-binding domains gives it distinct properties compared to VEGF-165 or other variants .

• Model system differences: Reconcile contradictions by critically evaluating the model systems used. Differences between cell lines, primary cells, zebrafish, and mouse models may all contribute to varying results. The transgenic heat-inducible VEGF zebrafish crossed with STAT3 knockout models provide valuable verification of mechanisms observed in cell culture .

• Technical approach integration: Combine multiple technical approaches (e.g., immunoprecipitation, GST pull-down, phosphorylation studies) to establish a consensus view of signaling mechanisms. For example, the interaction between VEGFR-2 and STAT3 has been confirmed through multiple complementary techniques .