VEGF Equine
Description
Tissue-Specific Expression and Exercise-Induced Regulation
Equine VEGF expression varies dynamically across tissues and physiological states:
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Baseline Expression: Highest in thyroid and lung tissues; lowest in skeletal muscle and appendix .
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Exercise Response: Blood leukocytes show a transient 60% increase in VEGFα mRNA 60 minutes post-exercise, likely due to hypoxia-induced HIF-1α activation (Figure 1) .
Figure 1: VEGFα Expression in Blood Leukocytes Post-Exercise
| Time Post-Exercise (min) | 0 | 30 | 60 | 90 | 120 |
|---|---|---|---|---|---|
| Relative Expression (Fold) | 1.0 | 1.2 | 1.6* | 1.4 | 1.1 |
| *Significant increase (p < 0.05) compared to baseline . |
Comparative Biology: Divergent Angiogenic Signaling in Horses
Equine endothelial cells (ECs) exhibit distinct responses to angiogenic stimuli compared to humans:
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VEGF-A vs. FGF2: Equine aortic ECs show minimal response to VEGF-A (≤50 ng/mL) but robustly proliferate and migrate in response to Fibroblast Growth Factor 2 (FGF2) .
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Receptor Expression: FGFR1 (FGF receptor) is predominant in equine ECs, whereas VEGFR2 (VEGF receptor) is more abundant in human ECs .
Therapeutic Applications of VEGF Modulation
Anti-VEGF therapies, validated in human medicine, are emerging in equine research:
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Bevacizumab (Avastin®):
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Platelet Lysates (ePL): Equine platelet lysates enhance VEGF-A secretion in arterial ring models, promoting vascular network formation (VEGF-A levels: 120 pg/mL with ePL vs. 80 pg/mL in controls) .
Table 2: Bevacizumab Efficacy in EqUVECs
| Concentration (mg/mL) | VEGF Reduction (%) | Cytotoxicity | Migration Inhibition (%) |
|---|---|---|---|
| 1 | 25 | None | 20 |
| 4 | 33 | None | 45 |
| 8 | 40 | Moderate | 65 |
Pathological Implications
Dysregulated VEGF signaling is implicated in equine diseases:
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Ocular Squamous Cell Carcinoma: VEGF overexpression correlates with tumor angiogenesis; anti-VEGF therapies may reduce recurrence post-surgery .
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Ethmoid Hematoma: VEGF is detectable in progressive ethmoid hematomas, suggesting a role in vascular lesion progression .
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Laminitis: Elevated VEGF levels disrupt hoof vascularization, though mechanistic studies remain limited .
Future Directions
Key research gaps include:
Product Specs
Vascular endothelial growth factor (VEGF) is a crucial signaling molecule for vasculogenesis and angiogenesis. Primarily known for its effects on vascular endothelial cells, VEGF also impacts other cell types, such as monocytes/macrophages, neurons, cancer cells, and kidney epithelial cells. Its roles include increasing vascular permeability, promoting angiogenesis, vasculogenesis, endothelial cell growth, cell migration, and inhibiting apoptosis. In vitro studies demonstrate its ability to stimulate endothelial cell mitogenesis and migration. As a vasodilator, VEGF enhances microvascular permeability and was initially termed vascular permeability factor. Elevated levels of VEGF are associated with POEMS syndrome (Crow-Fukase syndrome). Additionally, mutations in the VEGF gene are linked to proliferative and nonproliferative diabetic retinopathy.
Recombinant Equine VEGF, produced in E. coli, is a single, non-glycosylated polypeptide chain comprising 2 x 165 amino acids, resulting in a total molecular mass of 38.6 kDa. The purification process involves proprietary chromatographic techniques.
Lyophilized from a sterile (0.2 µm filtered) aqueous solution containing 10 mM Sodium Phosphate, pH 7.5.
For reconstitution, it is recommended to dissolve the lyophilized VEGF in sterile water to a concentration of 0.1 mg/ml. Further dilutions can be made in other aqueous solutions.
Lyophilized VEGF remains stable at room temperature for 3 weeks; however, storage in desiccated conditions below -18°C is recommended. Once reconstituted, Human VEGF should be stored at 4°C for 2-7 days. For long-term storage, freezing below -18°C is advised. It is highly recommended to add a carrier protein (0.1% HSA or BSA) for long-term storage. Avoid repeated freeze-thaw cycles.
The ED50, determined by HUVEC Proliferation assay, is ≤ 10 ng/mL, which corresponds to a specific activity of ≥ 1.0 x 105 units/mg.
VEGF-A, VPF, glioma-derived endothelial cell mitogen.
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Q&A
What is the fundamental role of VEGF in equine vascular biology?
VEGF (Vascular Endothelial Growth Factor) is a signaling protein that stimulates angiogenesis (formation of new blood vessels) in horses. While VEGF plays important roles in normal vascular development, recent research has revealed a fascinating species difference: unlike in humans where VEGF-A is the predominant angiogenic factor, equine endothelial cells respond much more strongly to Fibroblast Growth Factor 2 (FGF2) than to VEGF-A . This fundamental difference in growth factor responsiveness represents a critical consideration for researchers designing studies on equine vascular biology.
How is VEGF expression altered in equine pathological conditions?
In equine sarcoids, the most common skin tumors in horses, research has demonstrated strong and finely granular cytoplasmic VEGF staining in approximately 90% of keratinocytes, sarcoid fibroblasts, and endothelial cells . This overexpression suggests a dual role for VEGF in equine sarcoid development: not only increasing angiogenesis but also potentially controlling sarcoid fibroblast activity. Interestingly, despite strong VEGF expression, the resulting blood vessels often appear irregular in shape and lack distinct lumens, with microvessel area and perimeter measurements lower than normal vessels . This suggests that VEGF-induced vessels in sarcoids may be structurally immature, potentially creating a hypoxic microenvironment that further increases VEGF synthesis in a feedback loop .
What techniques are most effective for measuring equine VEGF levels?
Several validated methodologies exist for quantifying VEGF in equine samples:
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Enzyme-Linked Immunosorbent Assay (ELISA): Commercial horse-specific ELISA kits are available for detecting equine VEGF in plasma, serum, and tissue supernatants. Studies have successfully employed equine-specific ELISA kits from manufacturers such as MyBiosource (Cod. MBS035093) and Kingfisher Biotech . These assays typically involve sample incubation, antibody detection, and spectrophotometric measurement at 450 nm.
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Immunohistochemistry (IHC): This technique visualizes VEGF expression in tissue sections, identifying specific cell types expressing VEGF and assessment of expression patterns. Research on equine sarcoids has successfully employed IHC to demonstrate strong cytoplasmic VEGF staining .
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Western Blotting: Used to validate immunohistochemical findings regarding VEGF protein expression in equine tissues .
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PCR-Based Methods: Semi-quantitative real-time PCR allows assessment of VEGF gene expression in tissues such as the equine placenta .
What are the optimal protocols for isolating equine endothelial cells for VEGF studies?
Research has optimized specific protocols for isolating equine endothelial cells:
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Tissue source selection: The aorta provides a reliable source for isolating equine endothelial cells .
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Isolation methodology: Mechanical dissociation followed by magnetic purification using anti-VE-cadherin antibody results in endothelial cell-enriched cultures suitable for further study .
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Culture optimization: Endothelial growth medium (EGM) supplemented with horse serum (HS) provides effective conditions for maintaining equine endothelial cells .
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Verification of cell identity: Functional assessments including proliferation, migration (scratch assays), and tube formation capacity on extracellular matrix help confirm the endothelial phenotype .
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Molecular characterization: Gene expression analysis of endothelial markers and growth factor receptors (VEGFR1, VEGFR2, FGFR1) confirms cell identity and provides insights into functional properties .
How does the VEGF signaling pathway differ between equine and human endothelial cells?
Research has revealed remarkable species-specific differences in VEGF signaling:
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Receptor expression profiles: Unlike human endothelial cells, equine aortic endothelial cells (EAoECs) express FGFR1 at much higher levels than both VEGFR1 and VEGFR2, creating a fundamentally different receptor landscape for angiogenic signaling .
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Signal transduction: VEGF-A fails to promote ERK1/2 phosphorylation in equine endothelial cells, while it potently activates this pathway in human cells .
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Functional responses: While VEGF-A strongly stimulates proliferation, migration, and tube formation in human endothelial cells, it has minimal effects on these processes in equine endothelial cells .
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Alternative pathway predominance: Equine endothelial cells rely primarily on FGF2/FGFR1 signaling rather than VEGF-A/VEGFR signaling for angiogenic functions .
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Inhibitor sensitivity: Pharmacological inhibitors of FGFR1 (SU5402) or MEK (PD184352) effectively block FGF2-induced responses in equine endothelial cells, confirming the dependence on FGFR1/MEK-ERK signaling .
This discovery has significant implications for translational research and highlights the importance of species-specific considerations when studying vascular biology.
What quantitative differences exist in VEGF receptor expression between equine and human endothelial cells?
Gene expression analysis has revealed that equine aortic endothelial cells (EAoECs) express FGFR1 at significantly higher levels than both VEGFR1 and VEGFR2 . This contrasts sharply with human endothelial cells, which typically express high levels of VEGF receptors. This differential receptor expression profile explains the reduced sensitivity of equine endothelial cells to VEGF-A stimulation compared to human cells and their enhanced responsiveness to FGF2 . The Royal Veterinary College research explicitly states that "VEGF-A had far less of an effect on the cells of horses than those of humans. Instead, equine endothelial cells respond much more strongly to a different growth factor known as Fibroblast Growth Factor Two (FGF2)" .
How can equine arterial ring assays be optimized for studying VEGF-mediated angiogenesis?
The equine arterial ring assay provides an ex vivo model for studying angiogenesis with several key optimization parameters:
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Temporal considerations: Research has established the optimal window for analysis between days 5 and 9 after initial culture, based on the timeline of first sprouting (median = 4.47 days) and vascular regression (median = 10.73 days) .
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Growth media formulations:
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Alternative supplements:
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Quantification methodologies:
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VEGF-A monitoring: Collection of supernatants for VEGF-A concentration measurement using equine-specific ELISA provides insights into endogenous growth factor production .
What correlation exists between VEGF concentration and angiogenic parameters in ex vivo models?
Research using equine arterial ring models has found that VEGF-A concentration positively correlates with vascular network area (VNA) (P = .0243) . This suggests that despite the relative insensitivity of equine endothelial cells to VEGF-A, this growth factor still plays a role in equine angiogenesis. Notably, VEGF-A concentrations were significantly different between experimental groups (P = .0351), with higher levels in rings exposed to equine platelet lysate (ePL) at any concentration and horse serum (HS) . These findings indicate that blood-derived products like HS and ePL may stimulate the secretion of VEGF-A and serve as sources of this growth factor .
How does VEGF expression relate to microvessel morphology in equine sarcoids?
Research on equine sarcoids has revealed a complex relationship between VEGF expression and vascular structure:
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Increased but abnormal vasculature: Despite strong VEGF expression, blood vessels in sarcoids often appear irregular in shape and lack distinct lumens .
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Reduced vessel dimensions: Mean values of microvessel area and perimeter are lower than normal in sarcoid tissues, suggesting incomplete vascular maturation .
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Hypoxia hypothesis: Researchers suggest that the immature vasculature leads to a hypoxic microenvironment within the tumor, potentially further increasing VEGF synthesis in a positive feedback loop .
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Correlation with proliferation markers: VEGF overexpression occurs alongside moderate Ki67 positivity in 5-10% of dermal sarcoid fibroblasts, while Bcl-2 immunoreactivity was detected in 52% of sarcoid samples .
This suggests that VEGF in equine sarcoids promotes angiogenesis, but the resulting vessels are structurally and functionally abnormal, potentially contributing to tumor pathogenesis beyond simply increasing blood supply.
How is VEGF expression studied in equine perinatal development?
Research approaches to studying VEGF in equine perinatal development include:
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Multi-compartment analysis: Studies have evaluated VEGF levels at parturition from mare plasma, foal plasma, umbilical cord vein plasma, and amniotic fluid .
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Temporal profiling: Research has examined VEGF content in healthy foal plasma during the first 72 hours of life (at birth, 24 hours, and 72 hours post-birth) .
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Clinical correlations: Analyses have explored VEGF levels in relation to clinical parameters of mares and foals .
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Hormonal interactions: Studies have investigated relationships between VEGF and thyroid hormone levels (TT3 and TT4) in foals during early life .
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Placental expression: Research has assessed mRNA expression of VEGF and its receptors in the equine placenta .
| Sample Type | Collection Timepoint | Analysis Method | Parameter Measured |
|---|---|---|---|
| Mare plasma | Parturition | ELISA | VEGF concentration |
| Foal plasma | 0h, 24h, 72h after birth | ELISA | VEGF concentration |
| Umbilical cord vein plasma | Parturition | ELISA | VEGF concentration |
| Amniotic fluid | Parturition | ELISA | VEGF concentration |
| Placental tissue | Parturition | Semi-quantitative real-time PCR | VEGF mRNA expression |
What methodological limitations exist when studying VEGF-mediated angiogenesis in equine models?
Several technical challenges complicate equine VEGF research:
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Model variability: Equine arterial ring assays exhibit "a high degree of variability," making standardization difficult .
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Species-specific growth factor responses: The differential responsiveness to FGF2 versus VEGF-A necessitates adaptation of established human angiogenesis protocols for equine studies .
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Limited reagent availability: Researchers must carefully select appropriate equine-specific antibodies, growth factors, and ELISA kits .
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Culture optimization requirements: Different growth media formulations and supplements significantly impact angiogenic responses, requiring careful standardization .
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Quantification complexities: Image processing for quantifying vascular parameters requires specialized software and standardized protocols (e.g., Fiji image processing with specific settings) .
How should researchers approach experimental design when studying VEGF in equine models given species differences?
Based on current research findings, optimal experimental approaches include:
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Receptor profiling: Begin by characterizing VEGFR1, VEGFR2, and FGFR1 expression in the specific equine endothelial cell population being studied .
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Dual growth factor approach: Include both VEGF-A and FGF2 in experimental designs, with particular emphasis on FGF2 given its predominant role in equine angiogenesis .
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Signaling pathway validation: Confirm pathway activation through ERK1/2 phosphorylation assays and pharmacological inhibitor studies (e.g., FGFR1 inhibitor SU5402 or MEK inhibitor PD184352) .
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Functional readouts: Employ multiple functional assays (proliferation, migration, tube formation) to comprehensively assess angiogenic responses .
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Standardized supplements: Use well-characterized supplements such as horse serum (HS) or equine platelet lysate (ePL) at defined concentrations .
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VEGF measurement: Include VEGF-A concentration measurements in experimental protocols, as horse-derived supplements may stimulate endogenous VEGF-A production .
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Comparative approach: When possible, include parallel human endothelial cell experiments to highlight species differences and enhance translational relevance .
What are promising areas for further investigation of VEGF in equine models?
Based on current knowledge gaps, several promising research directions emerge:
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Molecular basis of species differences: Further investigation into the evolutionary and molecular basis for the differential growth factor responsiveness between equine and human endothelial cells.
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Pathological significance: Exploration of how the unique aspects of equine VEGF biology influence vascular diseases and wound healing in horses.
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Therapeutic implications: Development of FGF2-targeted rather than VEGF-targeted approaches for equine vascular disorders, based on the predominance of FGF2 responsiveness.
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Developmental regulation: More detailed characterization of VEGF expression and function during equine development, particularly in the perinatal period.
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Cross-talk mechanisms: Investigation of potential interactions between FGF2 and VEGF signaling pathways in equine endothelial cells.
How might understanding equine-specific VEGF biology inform comparative medicine?
The discovery of fundamental differences in angiogenic regulation between horses and humans has significant implications:
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Evolutionary insights: The predominance of FGF2 over VEGF-A responsiveness in equine endothelial cells suggests species-specific evolutionary adaptations in vascular regulation.
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Therapeutic development: Species-specific approaches may be required when developing pro- or anti-angiogenic therapies for horses versus humans.
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Model refinement: Understanding these differences will improve the design and interpretation of horse models used for human vascular disease research.
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Comparative physiology: These findings contribute to our broader understanding of species variations in fundamental biological processes.
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One Health applications: The knowledge gained from studying these differences may inform both veterinary and human medicine, advancing the One Health approach to biomedical research.
Product Science Overview
Vascular Endothelial Growth Factor (VEGF) is a critical signaling protein involved in both vasculogenesis (the formation of new blood vessels during embryonic development) and angiogenesis (the growth of blood vessels from pre-existing vasculature). VEGF is also known as Vascular Permeability Factor (VPF) or Vasculotropin due to its role in increasing vascular permeability and promoting endothelial cell growth .
VEGF is a homodimeric glycoprotein with a molecular weight ranging between 34 to 42 kDa. It is heparin-binding and exhibits potent angiogenic, mitogenic, and vascular permeability-enhancing activities specific to endothelial cells . VEGF belongs to the platelet-derived growth factor (PDGF) family of cystine-knot growth factors. The most significant member of this family is VEGF-A, which is the primary focus of most research and therapeutic applications .
VEGF exerts its effects by binding to specific receptor tyrosine kinases on the surface of endothelial cells. The two primary receptors are Flt-1 (fms-like tyrosine kinase) and KDR (kinase-insert-domain-containing receptor), also known as VEGFR-1 and VEGFR-2, respectively . These receptors have high affinity for VEGF and mediate its angiogenic and permeability-enhancing effects.
Recombinant Equine VEGF-A is derived from the expression of the equine VEGF-A gene in various host systems such as E. coli or Chinese Hamster Ovary (CHO) cells. The recombinant protein is purified using affinity chromatography techniques, ensuring high purity and activity . This recombinant form is used in various research applications, including cell culture, ELISA, and Western blotting .
VEGF plays a crucial role in both physiological and pathological processes. It is essential for wound healing, embryonic development, and the growth and metastasis of solid tumors. Elevated levels of VEGF have been observed in the synovial fluids of rheumatoid arthritis patients and in the sera of cancer patients . VEGF promotes the extravasation of plasma fibrinogen, leading to fibrin deposition, which alters the tumor extracellular matrix. This modified matrix subsequently promotes the migration of macrophages, fibroblasts, and endothelial cells .
Recombinant Equine VEGF-A is widely used in laboratory research to study angiogenesis, cell proliferation, and vascular permeability. It is also employed in various assays to quantify VEGF levels in biological samples . The protein’s activity is typically measured using a cell proliferation assay with human umbilical vein endothelial cells (HUVECs), where the effective dose (ED50) for this effect is usually between 1-5 ng/mL .
