VEGF Mouse

What is VEGF and what are its primary functions in mice?

VEGF is a signal protein produced by cells that stimulates vasculogenesis and angiogenesis. It serves as a critical mediator in restoring oxygen supply to tissues when blood circulation is inadequate, such as in hypoxic conditions . In mice, VEGF is crucial for embryonic development, as deletion of either a single or both alleles of the VEGF gene results in embryonic lethality by E9.5 and E10.5 with severe vascular abnormalities .

In adult mice, VEGF is expressed in virtually every tissue and is involved in mediating physiologic angiogenesis during the female reproductive cycle, wound healing, bone repair, and skeletal muscle response to exercise . It also plays essential roles in maintaining quiescent vasculature, as inhibition of VEGF or its receptors leads to alterations in microvasculature and vessel regression in multiple tissues including kidney, lung, pancreas, trachea, thyroid, and small intestine .

How can VEGF levels be accurately measured in mouse tissue samples?

VEGF levels in mouse samples can be quantified using Enzyme-Linked Immunosorbent Assay (ELISA) techniques. Commercial kits such as the Mouse VEGF ELISA Kit (KE10009) offer high sensitivity (2.0 pg/mL) and a detection range of 7.8-500 pg/mL specifically for mouse VEGF . These sandwich ELISA assays utilize antibodies specific for mouse VEGF that have been pre-coated onto microwells. The VEGF protein in samples is captured by the coated antibody after incubation, followed by detection using biotinylated secondary antibodies specific for mouse VEGF .

For optimal results when measuring VEGF in different sample types, validation studies report the following recovery rates:

Assay precision is also well-documented, with intra-assay coefficient of variation (CV) ranging from 3.4% to 8.6% and inter-assay CV ranging from 2.8% to 5.7% .

What are the main VEGF receptors in mice and how do they function?

VEGF signaling in mice is primarily mediated through two tyrosine kinase receptors: VEGFR1 (Flt1) and VEGFR2 (Flk1), both of which are essential for normal development . Homozygous mutation of either receptor results in embryonic lethality . Additionally, two co-receptors for VEGF, neuropilin-1 and neuropilin-2 (Nrp-1 and Nrp-2), are also required for proper embryonic development .

VEGFR2 is the primary receptor mediating VEGF's angiogenic effects in endothelial cells. Recent studies have demonstrated that VEGFR2 is expressed and activated in adult mouse tissues, providing evidence that VEGF plays an ongoing biological role in adult mice .

VEGFR1 plays a more complex role. While it is expressed by endothelial cells, its primary function appears to be modulating VEGFR2 signaling rather than directly mediating VEGF effects. This is supported by observations that mice with deletion of the intracellular kinase domain of VEGFR1 have no obvious vascular phenotype, while complete VEGFR1 disruption results in endothelial cell overgrowth and vascular disorganization . Recent evidence suggests that VEGFR1 can anchor VEGF to the cell surface, increasing its interaction with VEGFR2 .

What phenotypes result from VEGF gene manipulation in mice?

Genetic manipulation of VEGF in mice produces distinct phenotypes depending on the nature of the modification:

Complete knockout of VEGF is embryonically lethal. Deletion of either a single or both alleles of the VEGF gene in mice results in embryonic death by E9.5 and E10.5 with severe vascular abnormalities . This underscores the critical role of VEGF in normal development and the importance of proper VEGF expression regulation.

Interestingly, overexpression of VEGF also results in embryonic lethality, demonstrating that precise regulation of VEGF levels is essential for normal development .

In adult mice, tissue-specific VEGF overexpression produces distinct phenotypes. For example, cardiac-specific VEGF-B transgenic (TG) mice display normal contractile function but exhibit a distinct electrophysiological phenotype . This includes ECG changes such as increased QRSp time and decreased S and R amplitudes. At the cellular level, cardiomyocytes show decreased action potential upstroke velocity and increased action potential duration (APD 60-70) .

What are humanized VEGF mouse models and how are they used in research?

Humanized VEGF mouse models are engineered to express a humanized form of VEGF-A (hum-X VEGF) that can be recognized by anti-VEGF antibodies developed against human VEGF. These models were created using gene replacement technology to overcome the limitation that many anti-VEGF antibodies used in human therapy do not recognize mouse VEGF, making it difficult to evaluate their efficacy in standard mouse models .

The humanized VEGF (hum-X VEGF) expressed in these mouse models has biochemical and biological properties comparable to both wild-type mouse and human VEGF-A . This enables direct comparison of the pharmacological effects of different anti-VEGF antibodies in the same model system.

How does VEGF function in mouse models of lower limb ischemia and diabetes?

VEGF plays a therapeutic role in diabetic foot (DF) models, which represent one of the most serious complications of diabetes. Research using C57BL/6 mice fed a high-fat diet and injected with streptozocin to induce diabetic lower limb ischemia has shown that VEGF can alleviate lower limb ischemia .

The mechanism appears to involve VEGF-mediated skeletal muscle fiber type switching that promotes angiogenesis . Studies have measured VEGF protein levels and citrate synthase activity by ELISA, while muscle fiber type changes were assessed by immunofluorescence in the gastrocnemius muscle of these mice .

In experimental setups, adenovirus vectors expressing VEGF (Ad-VEGF-GFP) have been injected into the gastrocnemius muscle of diabetic mice with lower limb ischemia to study the therapeutic effects of VEGF delivery . This approach allows researchers to investigate not just the vascular effects of VEGF but also its impact on muscle fiber composition and metabolism.

What cellular mechanisms underlie VEGF's protective effects in adult mouse tissues?

VEGF is crucial for maintaining healthy vasculature in adult mice through several mechanisms:

Endothelial cell survival and maintenance: VEGF acts as a survival factor for endothelial cells in established blood vessels. When VEGF or its receptors are inhibited in adult mice, vessel regression occurs in multiple organs including the kidney, lung, pancreas, trachea, thyroid, and small intestine .

Fenestration maintenance: VEGF is involved in maintaining endothelial cell fenestrations, particularly in organs with fenestrated endothelium like the kidney glomeruli. Inhibition of VEGF leads to glomerular endotheliosis and proteinuria in mouse kidneys, resembling preeclampsia in humans .

Non-vascular cell effects: VEGF also affects non-endothelial cells in various tissues. In the lung, VEGF inhibition results in alveolar cell apoptosis and enlarged airspaces, suggesting that VEGF signaling is required for alveolar maintenance .

These protective mechanisms are particularly important when considering anti-VEGF therapies, as inhibition of VEGF in mice produces side effects similar to those observed in human patients treated with anti-VEGF agents, including hypertension and proteinuria .

How do different isoforms of VEGF produce distinct physiological effects in mice?

Different VEGF isoforms exhibit unique physiological effects in mice. VEGF-A is primarily known for its angiogenic effects and is critical for embryonic development . In contrast, VEGF-B has been identified as a potent mediator of vascular, metabolic, growth, and stress responses specifically in the heart .

Studies of cardiac-specific VEGF-B transgenic mice have shown that while VEGF-B overexpression does not affect contractile function, oxidative metabolism, or energy substrate preference in cardiomyocytes, it does induce a distinct electrophysiological phenotype . This phenotype is characterized by:

• ECG changes: Increased QRSp time and decreased S and R amplitudes

• Cellular changes: Decreased action potential upstroke velocity and increased action potential duration

• Ion channel alterations: Reduced expression of Nav1.5 leading to decreased sodium current (INa), and alterations in voltage-gated K+ currents including decreased density of transient outward current (Ito) and total K+ current (Ipeak)

• Transcriptional changes: Downregulation of Kv channel-interacting protein 2 (Kcnip2), a known modulatory subunit for Kv4.2/3 channel

These findings demonstrate that different VEGF isoforms can have specialized roles in specific tissues, beyond the canonical angiogenic functions typically associated with VEGF.

What are the methodological considerations when comparing anti-VEGF antibodies in humanized VEGF mouse models?

When comparing anti-VEGF antibodies in humanized VEGF mouse models, several methodological considerations are critical:

In vitro versus in vivo correlation: Research has shown that while in vitro studies consistently demonstrate a correlation between antibody binding affinity and potency at blocking VEGF-stimulated endothelial cell proliferation, this correlation does not reliably translate to in vivo efficacy in inhibiting tumor growth and angiogenesis . This discrepancy highlights the importance of in vivo validation rather than relying solely on in vitro potency.

Long-term safety assessment: Higher-affinity anti-VEGF antibodies have been associated with increased risk of glomerulosclerosis during long-term treatment . This suggests that safety evaluations should include extended treatment periods and thorough assessment of kidney function and histology.

Tumor versus host VEGF contributions: In standard mouse models, both tumor-derived and host-derived VEGF contribute to tumor vascularization, but many anti-VEGF antibodies only recognize human VEGF. Humanized VEGF mice overcome this limitation by allowing direct comparison of antibodies with different abilities to block host VEGF .

A comprehensive experimental approach should include:

• Comparison of antibody binding affinities using consistent assay methods

• Assessment of both in vitro and in vivo efficacy

• Evaluation of both anti-tumor efficacy and potential toxicity

• Long-term safety monitoring, particularly for renal effects

What are the most reliable methods for detecting VEGF expression patterns in mouse tissues?

Multiple complementary approaches can be used to reliably detect VEGF expression patterns in mouse tissues:

ELISA: Sandwich ELISA assays provide quantitative measurement of VEGF protein levels in tissue homogenates, serum, plasma, or cell culture supernatants. Commercial kits offer high sensitivity (as low as 2.0 pg/mL) and specificity for mouse VEGF .

For optimal reliability when using ELISA, internal validation is important. The following tables show typical performance metrics for mouse VEGF ELISA:

IntraAssay Precision:

InterAssay Precision:

Immunohistochemistry/Immunofluorescence: These techniques allow visualization of VEGF expression within the tissue context, providing information about the specific cell types expressing VEGF. This approach has been used to detect changes in skeletal muscle fiber types in relation to VEGF expression in models of diabetic lower limb ischemia .

RT-PCR/qPCR: These methods detect VEGF mRNA expression and can distinguish between different VEGF isoforms. In mouse models with altered VEGF expression, qPCR has been used to confirm changes in ion channel gene expression, such as the downregulation of Kv channel-interacting protein 2 (Kcnip2) in cardiac-specific VEGF-B transgenic mice .

How should researchers design experiments to study VEGF's role in tissue-specific contexts?

Designing experiments to study VEGF's role in tissue-specific contexts requires careful consideration of several factors:

• Selection of appropriate mouse models:

  • Tissue-specific transgenic models that overexpress VEGF in specific tissues (e.g., cardiac-specific VEGF-B TG mice for studying heart effects)

  • Conditional knockout models using Cre-loxP system to delete VEGF in specific cell types

  • Humanized VEGF models for studying anti-VEGF therapies

• Comprehensive functional analysis:

  • Tissue-specific functional assessments (e.g., cardiac function via echocardiography, electrophysiological recordings)

  • Cellular-level analyses (e.g., isolated cardiomyocyte function, ion channel recordings)

  • Molecular analyses (gene and protein expression)

• Translational relevance:

  • Disease models that mimic human conditions (e.g., diabetic lower limb ischemia model)

  • Comparison of findings in mouse models with human pathology

  • Therapeutic intervention studies (e.g., adenovirus-mediated VEGF delivery)

For example, in studying VEGF-B's effects on cardiac function, researchers conducted both in vivo and ex vivo analysis of cardiac-specific VEGF-B TG mice, examined oxidative metabolism and energy substrate preference in isolated cardiomyocytes, and performed detailed electrophysiological studies including ECG, action potential measurements, and ion current recordings . This multi-level approach provided comprehensive insights into the tissue-specific effects of VEGF-B overexpression.

What emerging technologies are advancing our understanding of VEGF biology in mouse models?

Several emerging technologies are enhancing VEGF research in mouse models:

• Single-cell RNA sequencing: This technology allows researchers to analyze VEGF expression and signaling at the single-cell level, providing unprecedented resolution of cell-specific responses to VEGF in heterogeneous tissues.

• Advanced genetic engineering: CRISPR/Cas9 technology enables more precise and efficient generation of VEGF knockout, knockin, and reporter mouse models, facilitating more sophisticated studies of VEGF biology.

• Intravital microscopy: Real-time visualization of VEGF-mediated angiogenesis in living mice allows dynamic assessment of vascular responses to VEGF manipulation.

• Adenoviral delivery systems: Advanced viral vectors allow targeted delivery of VEGF or VEGF inhibitors to specific tissues, as demonstrated in studies using Ad-VEGF-GFP in diabetic lower limb ischemia models .

• Multi-omics approaches: Integration of genomics, transcriptomics, proteomics, and metabolomics data provides a comprehensive view of how VEGF affects multiple biological processes simultaneously.

How can contradictory findings in VEGF mouse studies be reconciled?

Contradictory findings in VEGF mouse studies can often be attributed to several factors:

• Model-specific differences: Different mouse strains, genetic backgrounds, or model systems (e.g., knockout vs. overexpression) can produce seemingly contradictory results. For instance, while complete VEGF knockout is embryonically lethal, tissue-specific VEGF manipulations may show varying phenotypes depending on the tissue context .

• Developmental timing: The effects of VEGF manipulation may differ dramatically depending on whether it occurs during embryonic development, postnatal growth, or adulthood .

• Isoform-specific effects: Different VEGF isoforms (VEGF-A, VEGF-B, etc.) have distinct biological effects. For example, VEGF-B affects cardiac electrophysiology but not contractile function or metabolism , while VEGF-A is critical for angiogenesis.

• Dose-dependent effects: Both insufficient and excessive VEGF levels can be detrimental, as demonstrated by the embryonic lethality of both VEGF knockout and overexpression .

• Context-dependent signaling: VEGF effects may vary based on the physiological or pathological context. For instance, the humanized VEGF mouse model revealed that while higher-affinity anti-VEGF antibodies showed greater potency in vitro, this did not consistently translate to greater in vivo efficacy in tumor models, despite causing more significant side effects .

To reconcile contradictory findings, researchers should:

• Clearly define the specific VEGF isoform being studied

• Consider the temporal and spatial context of VEGF manipulation

• Use multiple complementary approaches to validate findings

• Directly compare different models under identical experimental conditions

• Carefully consider physiological relevance of the model systems used