VEGF E (Orf Virus)

What is VEGF-E and how does it relate to Orf virus pathology?

VEGF-E is a novel member of the vascular endothelial growth factor family encoded by the parapoxvirus Orf virus. It carries the characteristic cysteine knot motif present in all mammalian VEGFs while forming a microheterogenic group distinct from previously described VEGF family members .

Orf virus causes pustular dermatitis in sheep, goats, and humans, with lesions histopathologically characterized by massive capillary proliferation, dilation, and dermal swelling . These vascular changes are directly attributable to VEGF-E expression, as deletion of the VEGF-E gene results in substantially reduced lesion development in experimentally infected sheep when compared with the parental virus strain . The viral lesions also exhibit extensive epidermal hyperplasia and rete ridge formation, which research has shown can be induced by VEGF-E independently of its vascular effects .

How does VEGF-E differ structurally and functionally from mammalian VEGFs?

What bioassays demonstrate VEGF-E's functional activity?

VEGF-E exhibits numerous bioactivities comparable to VEGF-A in various experimental systems:

• Endothelial cell proliferation: VEGF-E stimulates the proliferation of human umbilical vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HDMVECs) but not normal human dermal fibroblasts (NHDFs) or human smooth muscle cells (HSMCs) .

• Tissue factor production: Incubation of endothelial cells with 6 ng/ml VEGF-E for 6 hours results in significant production of tissue factor (60 pg/ml compared to 2 pg/ml in controls), similar to VEGF-A .

• Chemotactic migration: VEGF-E induces directional migration of endothelial cells in modified Boyden chamber assays .

• Endothelial sprouting: In three-dimensional collagen gel assays, VEGF-E stimulates endothelial cell sprouting, mimicking early stages of angiogenesis .

• Vascular permeability: Purified VEGF-E from Orf virus strain NZ2 induces vascular permeability similar to VEGF-A .

• Keratinocyte function: VEGF-E directly induces keratinocyte migration and proliferation through VEGFR-2, promoting epidermal regeneration .

How is VEGF-E produced for experimental studies?

Researchers have employed two main approaches to produce VEGF-E for experimental use:

Mammalian expression system:
VEGF-E can be expressed as a native protein in mammalian cells by cloning the VEGF-E gene into a mammalian expression vector (e.g., pCB6-Bam) under control of a strong promoter (e.g., cytomegalovirus promoter) . When transfected into COS-7 cells, the protein is efficiently secreted into the culture medium and exhibits mitogenic activity on endothelial cells .

Bacterial expression system:
For larger quantities, recombinant VEGF-E can be produced in Escherichia coli with a hexa-histidine tag to facilitate purification . The tag can be proteolytically removed if needed, and the purified recombinant protein demonstrates similar biological activity to the native form .

What in vivo models are appropriate for studying VEGF-E functions?

Several in vivo models have proven effective for investigating VEGF-E's biological activities:

• Mouse skin models: Injection of VEGF-E into normal mouse skin allows researchers to quantify increases in endothelial cell numbers, blood vessel formation, and epidermal thickening .

• Wound healing models: VEGF-E can be introduced into experimental skin wounds to study its effects on neo-epidermal thickness, rete ridge formation, and re-epithelialization kinetics .

• Viral pathogenesis models: Comparing wild-type Orf virus with VEGF-E deletion mutants in experimental infections of sheep provides insights into VEGF-E's contribution to viral pathology .

How does VEGF-E signaling differ from other VEGF family members?

VEGF-E's unique receptor binding profile results in distinctive signaling characteristics:

• Receptor autophosphorylation: VEGF-E binding to VEGFR-2 results in receptor autophosphorylation, initiating downstream signaling cascades .

• Calcium mobilization: VEGF-E induces a biphasic rise in free intracellular Ca²⁺ concentration in endothelial cells, similar to VEGF-A .

• VEGFR-2-specific effects: Because VEGF-E activates VEGFR-2 without VEGFR-1 engagement, it allows researchers to isolate VEGFR-2-specific signaling events from those requiring VEGFR-1 activation .

• Metalloproteinase induction: In wound models, VEGF-E increases matrix metalloproteinase (MMP)-2 and MMP-9 expression, which contributes to extracellular matrix remodeling during tissue repair .

What techniques can assess VEGF-E's effects on keratinocyte function?

Recent research has revealed VEGF-E's ability to regulate keratinocyte function independently of its angiogenic effects. Methods to study these effects include:

• Keratinocyte proliferation assays: Direct measurement of cell proliferation in response to VEGF-E treatment, with VEGFR-2 neutralizing antibodies as controls to confirm receptor specificity .

• Migration assays: Quantification of keratinocyte migration in scratch or Boyden chamber assays following VEGF-E stimulation .

• Quantitative RT-PCR: Analysis of gene expression changes in keratinocytes or wound tissue after VEGF-E treatment, particularly focusing on matrix metalloproteinases and growth factors .

• Histological analysis: Assessment of epidermal thickness, rete ridge formation, and keratinocyte numbers in skin sections following VEGF-E administration .

How does gene deletion help elucidate VEGF-E's role in viral pathogenesis?

Recombinant DNA techniques allow creation of Orf virus variants lacking the VEGF-E gene, providing a powerful approach to understand its role in viral pathology:

• Comparative infection studies: Experiments show that deletion of VEGF-E does not affect virus growth in tissue culture but significantly reduces lesion development in experimentally infected sheep .

• Supernatant activity testing: Supernatants from cells infected with wild-type Orf virus stimulate endothelial cell proliferation, while those from VEGF-E deletion mutant infections do not, confirming VEGF-E as the factor responsible for this activity .

• Complementation analysis: Reintroduction of the VEGF-E gene into deletion mutants can restore pathogenic potential, confirming the specific contribution of this viral factor to disease pathology.

What insights does VEGF-E provide into VEGF receptor biology?

VEGF-E serves as a valuable tool for dissecting receptor-specific functions within the VEGF signaling system:

• VEGFR-2 sufficiency: The potent angiogenic activity of VEGF-E despite its inability to bind VEGFR-1 demonstrates that VEGFR-2 activation alone is sufficient to efficiently stimulate angiogenesis .

• Receptor cooperation: By comparing cellular responses to VEGF-E (VEGFR-2 specific) versus VEGF-A (binds both VEGFR-1 and VEGFR-2), researchers can identify processes requiring cooperative receptor activation.

• Neuropilin-1 function: VEGF-E binding to neuropilin-1 provides opportunities to study this co-receptor's contribution to VEGFR-2 signaling in various biological contexts .

What potential therapeutic applications might emerge from VEGF-E research?

VEGF-E's unique properties suggest several potential therapeutic applications:

• Wound healing: VEGF-E enhances wound re-epithelialization, increases neo-epidermal thickness, and promotes rete ridge formation while simultaneously stimulating angiogenesis . These properties could be valuable for developing treatments for chronic wounds or burns.

• Selective angiogenesis modulation: VEGF-E's ability to induce angiogenesis through VEGFR-2 alone might offer advantages over VEGF-A by avoiding unwanted effects mediated through VEGFR-1, potentially resulting in more targeted therapies for ischemic conditions.

• Epidermal regeneration: VEGF-E's direct effects on keratinocytes could be exploited for skin regeneration applications in dermatological conditions characterized by compromised epithelial integrity or function.

How might comparative studies between different VEGF-E isolates inform structure-function relationships?

VEGF-E sequences from different Orf virus strains form a microheterogenic group with some sequence diversity . Comparing the biological activities of these natural variants could:

• Identify key structural determinants of receptor binding specificity

• Map domains responsible for different biological activities

• Provide insights into the evolutionary relationship between viral and mammalian VEGFs

• Guide rational design of modified VEGF variants with tailored receptor selectivity and biological functions

What methodological issues must be addressed when studying VEGF-E?

Researchers face several technical challenges when investigating VEGF-E:

• Protein production: Ensuring proper folding and disulfide bond formation in recombinant VEGF-E to maintain biological activity requires careful optimization of expression systems and purification protocols.

• Distinguishing direct and indirect effects: In complex biological systems, differentiating VEGF-E's direct effects from secondary consequences requires appropriate controls, including receptor-neutralizing antibodies .

• Species considerations: When using animal models, researchers must account for potential species-specific differences in VEGF receptor expression and function.

• Viral context: Understanding how VEGF-E functions within the broader context of Orf virus infection requires consideration of other viral factors that may influence its activity.

How can researchers assess the contribution of VEGF-E to complex biological processes?

Studying VEGF-E's role in complex processes like wound healing or viral pathogenesis requires multifaceted approaches:

• Genetic approaches: Use of VEGF-E deletion mutants or conditional expression systems to manipulate VEGF-E levels in specific contexts.

• Receptor knockdown/knockout: Silencing or deletion of specific receptors (VEGFR-2, neuropilin-1) to confirm their involvement in VEGF-E-mediated effects.

• Pathway inhibitors: Selective blocking of downstream signaling components to map the intracellular mechanisms mediating VEGF-E's diverse biological effects.

• Combinatorial studies: Comparing effects of VEGF-E alone versus combinations with other growth factors to understand synergistic or antagonistic interactions.