VEGF Rat, His

What are the key structural differences between rat VEGF isoforms?

Rat VEGF exists in multiple isoforms, with VEGF164 being predominant in research settings. The rat VEGF164 protein consists of amino acids Ala27-Arg190 and features different binding properties compared to other isoforms. Western blot analyses show that VEGF164 can be detected as both homodimeric and monomeric forms at approximately 54 kDa and 24 kDa, respectively, under reducing conditions . The structural variations between isoforms directly impact their biological activities, with VEGF164 showing higher binding affinity to VEGFR2/Flk1 receptors compared to shorter isoforms.

How does rat VEGF164 compare functionally to human VEGF165?

While structurally similar, rat VEGF164 and human VEGF165 exhibit important species-specific differences. Cross-reactivity studies using direct ELISAs demonstrate approximately 20% cross-reactivity between anti-rat VEGF antibodies and recombinant human VEGF165 and VEGF121 . Despite this partial cross-reactivity, functional studies show that rat VEGF164 has an ED50 of 0.75-3.75 ng/mL for stimulating proliferation in human umbilical vein endothelial cells (HUVECs) , which differs from human VEGF165. These differences must be considered when designing cross-species experiments or when translating findings from rat models to human applications.

What is the significance of the VEGF/Flk1 pathway in rat models?

The VEGF/Flk1 (VEGFR2) pathway plays a crucial role in rat angiogenesis and endothelial cell function. Research indicates that this pathway mediates roxarsone promotion of rat vascular endothelial cell proliferation, migration, and tube-like formation in vitro, as well as tumor growth and angiogenesis in mouse xenograft models . Blocking experiments using Flk1 antibodies significantly reduce cell viability compared to Flt1 (VEGFR1) antibody treatments (p<0.01), indicating the primary importance of VEGFR2/Flk1 signaling . Small interfering RNA (siRNA) targeting Flk1 significantly attenuates these promotion effects, confirming the mechanistic importance of this receptor in VEGF-mediated processes.

What are the optimal conditions for detecting rat VEGF164 in Western blot applications?

For optimal Western blot detection of rat VEGF164, the following protocol yields consistent results:

• Use PVDF membrane for protein transfer

• Probe with 0.1 μg/mL of anti-rat VEGF164 antibody (e.g., Goat Anti-Rat VEGF Antigen Affinity-purified Polyclonal Antibody)

• Follow with HRP-conjugated secondary antibody

• Conduct experiments under reducing conditions using appropriate buffer systems

Table 1: Comparison of Detection Methods for Rat VEGF164

Note that this antibody typically does not detect natural VEGF in lysates from cell lines or tissues, which is an important limitation to consider when designing experiments.

How can researchers optimize immunohistochemical detection of rat VEGF in tissue sections?

For optimal immunohistochemical detection of rat VEGF in tissue sections:

• Use perfusion-fixed frozen sections rather than paraffin-embedded samples for better epitope preservation

• Apply 15 μg/mL of anti-rat VEGF164 antibody and incubate overnight at 4°C

• Utilize appropriate HRP-DAB staining systems with hematoxylin counterstaining

This methodology has been successfully employed to detect VEGF164 in rat kidney tissues, revealing specific expression patterns in the renal microvasculature and tubular epithelial cells. For quantitative assessment of expression, semiquantitative analysis using standardized scoring systems has been validated in multiple studies examining VEGF expression in models of kidney regeneration and pathology .

What are the most reliable methods to assess VEGF functionality in rat cell culture systems?

To reliably assess VEGF functionality in rat cell systems, the cell proliferation assay using HUVECs represents the gold standard approach:

• Treat HUVEC cells with varying concentrations of recombinant rat VEGF164 (typically 0-50 ng/mL)

• Measure proliferation after 24-72 hours using appropriate proliferation assays

• For neutralization studies, include anti-VEGF antibodies at concentrations of 0.2-0.6 μg/mL in the presence of 20 ng/mL rat VEGF164

The effective dose for 50% stimulation (ED50) typically falls between 0.75-3.75 ng/mL for rat VEGF164 . Additional functional assays include endothelial cell migration (Boyden chamber assay) and tube formation on Matrigel, both of which provide complementary information about VEGF's angiogenic properties.

How does His-tagging affect rat VEGF protein activity and stability?

When using His-tagged rat VEGF in functional assays, it is advisable to:

• Compare activity with non-tagged variants in dose-response experiments

• Verify receptor binding using surface plasmon resonance or other binding assays

• Assess dimerization status, as proper VEGF dimerization is essential for biological activity

What are the optimal purification strategies for His-tagged rat VEGF?

For optimal purification of His-tagged rat VEGF:

• Express the protein in appropriate expression systems (mammalian cells preferred for proper glycosylation)

• Utilize immobilized metal affinity chromatography (IMAC) with Ni-NTA or Co-based resins

• Apply a step-gradient elution protocol with imidazole (20-250 mM)

• Include additional purification steps such as size exclusion chromatography to ensure dimeric VEGF isolation

• Verify purity by SDS-PAGE under both reducing and non-reducing conditions

Table 2: Purification Strategy Comparison for His-Tagged Rat VEGF

The purified protein should be validated for endotoxin levels (<0.1 EU/μg protein) and bioactivity in HUVEC proliferation assays before use in critical experiments.

How does rat VEGF expression change in models of ischemia and what mechanisms are involved?

In rat models of ischemia, VEGF expression undergoes significant temporal and spatial changes. Studies of brain ischemia have shown that Vegfa mRNA is upregulated in both cortical and subcortical regions of the ipsilateral (ischemic) hemisphere compared to the contralateral side . This upregulation is associated with increased vascular permeability, as evidenced by Evans blue extravasation studies.

Mechanistically, the ischemia-induced VEGF expression is mediated by:

• Hypoxia-inducible factor 1α (HIF-1α) activation

• Brain-associated macrophages (BAMs), as their depletion attenuates ischemia-induced Vegfa mRNA expression

• Inflammatory cytokine signaling cascades

Western blot analyses reveal that VEGF164 can be detected as both homodimeric and monomeric forms at approximately 54 kDa and 24 kDa in ischemic brain tissue . Targeting these mechanisms provides potential therapeutic opportunities for modulating post-ischemic angiogenesis and vascular repair.

What is the role of VEGF in rat kidney regeneration models and how can it be therapeutically modulated?

VEGF plays a crucial role in kidney regeneration in rat models. Studies using male Munich-Wistar Frömter (MWF) rats have demonstrated that metanephroi (MET) transplantation significantly increases VEGF expression in renal tissues compared to saline controls, as shown by both Real-Time RT-PCR and immunohistochemistry . This increased expression correlates with improved renal function and structure.

Key findings from these studies include:

• Upregulation of multiple growth factors (VEGF, FGF2, HGF, IGF-1) following MET transplantation

• Sex-specific differences in VEGF expression patterns

• Correlation between VEGF levels and functional recovery measures

Therapeutic modulation strategies include:

• Direct administration of recombinant rat VEGF164

• Transplantation of cells engineered to overexpress VEGF

• Use of compounds that stabilize HIF-1α to indirectly increase VEGF production

• Targeted delivery systems to maximize local effects while minimizing systemic exposure

How does VEGF signaling interact with inflammatory processes in rat models of disease?

VEGF signaling and inflammatory processes exhibit complex bidirectional interactions in rat disease models. Research demonstrates that VEGF can both promote and inhibit inflammation depending on the context, timing, and dosage. In spinal cord injury models, VEGF acts as a key mediator leading to different recovery levels in neonatal versus adult rats by regulating inflammatory responses, protecting damaged neurons, and promoting reestablishment of spinal neural circuits .

Evidence suggests that:

• Low-dose VEGF treatment can reduce inflammatory cytokine production

• VEGF protects neurons from inflammation-induced apoptosis

• The VEGF/Flk1 pathway modulates microglial activation and phenotype switching

Importantly, the anti-inflammatory effects of VEGF appear to be receptor-specific, with Flk1 (VEGFR2) signaling mediating protective effects while Flt1 (VEGFR1) may promote inflammatory responses in certain contexts. These findings highlight the potential for receptor-specific targeting approaches in therapeutic development.

What strategies can overcome challenges in studying VEGF in complex rat tissue microenvironments?

Studying VEGF in complex rat tissue microenvironments presents several challenges that can be addressed through:

• Multi-scale imaging approaches:

  • Combine immunohistochemistry with high-resolution confocal microscopy

  • Implement tissue clearing techniques (CLARITY, iDISCO) for 3D visualization

  • Use multiplexed immunofluorescence to simultaneously visualize VEGF, receptors, and cellular markers

• Cell-specific analyses:

  • Employ laser capture microdissection to isolate specific cell populations

  • Utilize single-cell RNA sequencing to characterize heterogeneous VEGF expression

  • Develop cell type-specific conditional expression systems

• Dynamic monitoring:

  • Implement in vivo microscopy with fluorescently labeled VEGF variants

  • Use DCE-MRI to monitor vascular responses to VEGF in real-time

  • Develop biosensor systems for continuous VEGF activity monitoring

These approaches have successfully addressed the limitations of conventional techniques in capturing the dynamic and spatially complex nature of VEGF signaling in various rat disease models.

How can researchers address contradictory findings regarding VEGF effects in different rat experimental models?

Contradictory findings regarding VEGF effects across different rat experimental models can be reconciled through:

• Systematic comparison of experimental variables:

  • Standardize dose, timing, and duration of VEGF administration

  • Consider rat strain differences (Sprague-Dawley vs. Wistar vs. specialized disease models)

  • Account for age and sex differences in VEGF responsiveness

• Receptor-specific analysis:

  • Differentiate between Flk1 (VEGFR2) and Flt1 (VEGFR1) mediated effects

  • Consider co-receptor (neuropilins, heparan sulfate proteoglycans) involvement

  • Analyze isoform-specific effects (VEGF120 vs. VEGF164 vs. VEGF188)

• Context-dependent interpretation:

  • Recognize that VEGF effects may be biphasic (beneficial at low doses, detrimental at high doses)

  • Consider tissue microenvironment differences (inflammatory status, hypoxia levels)

  • Integrate temporal dynamics into experimental design and analysis

For example, studies have shown that red wine polyphenols exert dose-dependent effects on VEGF-mediated angiogenesis in rats, with low doses being proangiogenic and high doses anti-angiogenic , illustrating the importance of dosage considerations in experimental design.

What novel methodologies are emerging for studying VEGF signaling dynamics in rat models?

Emerging methodologies for studying VEGF signaling dynamics in rat models include:

• Advanced imaging techniques:

  • Dynamic contrast-enhanced (DCE) and diffusion-weighted (DW) MRI for non-invasive monitoring of vascular responses to anti-VEGF treatments in rat glioma models

  • Intravital multiphoton microscopy for real-time visualization of VEGF-induced vascular changes

  • Super-resolution microscopy for nanoscale receptor clustering analysis

• Genetic engineering approaches:

  • CRISPR/Cas9-mediated gene editing for isoform-specific VEGF modifications

  • Optogenetic control of VEGF release for precise spatiotemporal signaling studies

  • Conditional knockout systems for cell-specific VEGF or receptor deletion

• High-throughput analytical methods:

  • Phosphoproteomics to map complete VEGF signaling networks

  • Spatial transcriptomics to visualize VEGF expression patterns while preserving tissue architecture

  • AI-assisted image analysis for quantifying complex vascular patterns

These technologies are enabling unprecedented insights into the spatial and temporal dynamics of VEGF signaling in physiological and pathological conditions, moving beyond static endpoint measurements to capture the dynamic nature of angiogenic processes.