EGFL7 Antibody

What is EGFL7 and what are its primary biological functions?

EGFL7 is an extracellular matrix (ECM)-associated protein primarily expressed in activated endothelium. It plays critical roles in regulating vascular development and homeostasis. The protein supports endothelial cell adhesion to the ECM and protects endothelial cells from stress-induced apoptosis . EGFL7 regulates vascular tubulogenesis in vivo, inhibits platelet-derived growth factor (PDGF)-BB-induced smooth muscle cell migration, and promotes angiogenesis .

While EGFL7 is highly expressed during vascular development, it is typically downregulated in adult endothelium except during tissue repair or regeneration. Elevated expression has been detected in several tumor types, including colon, gastric, breast, kidney, liver, and brain tumors .

How do anti-EGFL7 antibodies affect endothelial cell function?

Anti-EGFL7 antibodies block the adhesive and prosurvival activities of EGFL7 in vitro. Specifically, they inhibit EGFL7's ability to protect endothelial cells from stress conditions. When administered in vivo, anti-EGFL7 antibodies enhance the anti-angiogenic activity and survival benefits resulting from VEGF blockade in various cancer models . Treatment with anti-EGFL7 antibodies reduces tumor vascularization by targeting several aspects of vessel maturation, including pericyte coverage, smooth muscle cell recruitment, and basement membrane formation .

What are the expression patterns of EGFL7 in normal versus pathological tissues?

In healthy adults, EGFL7 expression is relatively low and primarily restricted to the vasculature. During embryonic development, EGFL7 is highly expressed in developing blood vessels. In pathological conditions such as glioblastoma, immunohistochemical analysis reveals that blood vessels (particularly large vessels with a distinct lumen) stain strongly positive for EGFL7, while tumor cells themselves typically do not express significant levels . Quantification of EGFL7 staining in glioma specimens shows approximately 25-40% positive intratumoral blood vessels in each specimen .

How can researchers validate anti-EGFL7 antibody specificity?

When validating anti-EGFL7 antibodies, researchers should employ multiple approaches:

• Western blotting: Compare expression in known positive (placenta, HUVEC) and negative (Jurkat cells) controls. The expected molecular weight is consistent with literature reports (PMID: 18497746) .

• Immunostaining: Perform co-staining with alternative anti-EGFL7 antibodies from different sources to confirm specificity, as demonstrated in glioma specimens where different antibodies showed 100% overlap .

• Genetic controls: Use EGFL7 knockout models, noting that EGFL7 KO mice generated by retroviral gene trap insertion may have reduced miR-126 expression (~80% reduction), whereas newer models (EGFL7fl/fl;Cdh5-CreERT2) allow specific deletion without affecting miR-126 .

• Recombinant protein blocking: Pre-incubation with recombinant EGFL7 should abolish specific antibody staining.

What experimental models are suitable for studying EGFL7 function?

Several validated model systems have been used to study EGFL7 function:

• Cell culture models: HUVECs are widely used for studying EGFL7's effects on endothelial cell adhesion, migration, and survival under stress conditions .

• Tumor xenograft models: Human U87 glioblastoma cells xenografted into nude mice have been used to evaluate EGFL7's role in tumor vascularization .

• Syngeneic tumor models: GL261 glioma cells implanted into C57BL/6 mice provide an immunocompetent model for studying EGFL7's effects on tumor growth and vascularization .

• Genetic models: Several EGFL7 knockout mice are available:

  • Constitutive EGFL7 KO (with reduced miR-126)

  • Conditional EGFL7fl/fl;Cdh5-CreERT2 (endothelial-specific deletion without affecting miR-126)

  • Constitutive miR-126 KO (without affecting EGFL7)

• Disease-specific models: Experimental autoimmune encephalomyelitis (EAE) for multiple sclerosis and murine models of acute graft-versus-host disease (aGVHD) following allogeneic hematopoietic stem cell transplantation .

How can circulating progenitor cells be used as biomarkers for anti-EGFL7 activity?

Circulating progenitor cells (CPCs) have been identified as a pharmacodynamic marker for interrogating in vivo activities of anti-EGFL7. These cells are characterized as CD34hiCD31dimCD45dim and also express markers of progenitor cells, including CD133, CD117, and Aldha1 .

To use CPCs as biomarkers:

• Isolation protocol: Peripheral blood mononuclear cells are isolated and analyzed by flow cytometry for the CD34hiCD31dimCD45dim phenotype.

• Quantification: The number of CPCs is determined by enumeration via flow cytometry.

• Response assessment: Treatment with anti-EGFL7 results in a reduction in the number of CPCs compared to control groups in both tumor-bearing animals and cancer patients from phase I clinical trials .

• Dose-response relationship: The degree of CPC reduction correlates with anti-EGFL7 dosing, making this a useful pharmacodynamic marker for dose selection in clinical development .

How does EGFL7 affect tumor growth and vascularization in glioblastoma models?

EGFL7 significantly impacts glioblastoma growth and vasculature development as evidenced by several experimental findings:

• Tumor size: GL261 glioma cells engineered to ectopically express human or murine EGFL7 produced tumors more than three times larger than control tumors when implanted in mice .

• Survival impact: Mice bearing EGFL7-positive tumors died significantly earlier compared to control groups. The median survival time for mice with gliomas expressing human or murine EGFL7 was 29.5 days or 31.5 days, respectively, compared to 34 days in the control group .

• Vascular density: Blood vessel analysis using the endothelial marker CD31 demonstrated significantly higher microvascular density in tumors expressing EGFL7 .

• Vessel maturation: EGFL7 expression increased blood vessel maturation as measured by:

  • Increased pericyte coverage (PDGFRβ+)

  • Enhanced smooth muscle cell recruitment (SMA+)

  • Improved basement membrane formation (Col IV+)

• Vascular integrity: T1-weighted MRI analysis showed less contrast agent (Gadovist) leakage into the glioma mass in EGFL7-expressing tumors, suggesting increased vessel maturation and integrity .

What mechanisms explain EGFL7's effects on tumor vasculature?

EGFL7 affects tumor vasculature through several molecular mechanisms:

• Integrin regulation: EGFL7 increases surface expression of integrin α5β1 on endothelial cells. This was demonstrated through:

  • Biotin labeling of surface-bound integrins

  • Flow cytometry analysis showing increased surface levels of α5β1 after EGFL7 treatment

• Integrin-fibronectin interactions: EGFL7 and fibronectin (Fn) act additively to increase surface expression of integrin α5β1, enhancing endothelial cell adhesion to the extracellular matrix .

• αVβ3 integrin trafficking: EGFL7 binds integrin αVβ3, which affects the intracellular trafficking of α5β1, resulting in increased surface expression of both integrins .

Table 1: EGFL7 effects on integrin surface expression in endothelial cells

How does anti-EGFL7 antibody treatment affect tumor vasculature?

Anti-EGFL7 antibody treatment has significant effects on tumor vasculature:

• Reduced vascularization: CD31 staining was significantly lower in groups treated with anti-EGFL7, anti-VEGF, or a combination of both blocking antibodies compared to control .

• Vessel maturation markers: Treatment effects on vessel maturation varied by marker:

  • PDGFRβ (pericytes): Individual blocking of EGFL7 or VEGF did not significantly influence pericyte coverage, but combinational treatment significantly decreased PDGFRβ-positive pericytes .

  • SMA (smooth muscle cells): Both anti-EGFL7 and anti-VEGF significantly reduced smooth muscle cell coverage, with the combination therapy showing the strongest effect .

  • Col IV (basement membrane): Both antibodies reduced Col IV expression, with anti-EGFL7 showing stronger effects than anti-VEGF and the combination exhibiting the greatest decrease .

• Synergy with anti-VEGF: Anti-EGFL7 enhanced both the anti-angiogenic activity and survival benefits resulting from VEGF blockade in xenograft tumor models and genetically engineered mouse models of cancer .

What is the role of EGFL7 in multiple sclerosis and neuroinflammation?

EGFL7 has an important role in central nervous system (CNS) inflammation, particularly in multiple sclerosis (MS):

• Expression patterns: EGFL7 expression is increased in the CNS vasculature of patients with MS and in mice with experimental autoimmune encephalomyelitis (EAE), a model of MS .

• Cellular localization: Perivascular CD4 T lymphocytes colocalize with ECM-bound EGFL7 in MS lesions .

• Immune cell interaction: Human and mouse activated T cells upregulate EGFL7 ligand αvβ3 integrin and can adhere to EGFL7 through this integrin .

• Impact in knockout models:

  • EGFL7-knockout mice show earlier onset of EAE and increased brain and spinal cord parenchymal infiltration of T lymphocytes

  • Endothelial cell-restricted EGFL7-KO is associated with similar EAE worsening

• Therapeutic potential: Treatment with recombinant EGFL7 improves EAE, reduces expression of melanoma cell adhesion molecule (MCAM), and tightens the blood-brain barrier in mouse models .

How does EGFL7 influence acute graft-versus-host disease after stem cell transplantation?

EGFL7 plays a significant role in acute graft-versus-host disease (aGVHD) following allogeneic hematopoietic stem cell transplantation:

• Therapeutic effect: Treatment with recombinant EGFL7 (rEGFL7) in two different murine models of aGVHD decreased disease severity and improved survival in recipient mice compared to controls .

• Mechanism: EGFL7 decreases inflammation by repressing endothelial cell activation and T-cell migration .

• Immune reconstitution: rEGFL7 treatment resulted in higher thymocyte, T cell, B cell, and dendritic cell counts in recipient mice after allogeneic transplantation .

• Maintained anti-tumor activity: Importantly, rEGFL7 treatment did not inhibit the graft-versus-leukemia effect, suggesting it could reduce GVHD while preserving the therapeutic anti-cancer benefits of the transplantation .

How does EGFL7 interact with NOTCH signaling pathways?

EGFL7 has important interactions with the NOTCH signaling pathway, particularly in acute myeloid leukemia (AML):

• Protein binding: Using an antibody interaction array, EGFL7 was found to bind directly to several signaling proteins expressed by primary AML blasts, including NOTCH receptors .

• Signaling antagonism: EGFL7 antagonizes canonical NOTCH ligand binding in AML cells:

  • Stimulation of AML blasts with recombinant EGFL7 reduced NOTCH intracellular domain and NOTCH target gene expression

  • Recombinant EGFL7 inhibits DELTA-like (DLL) 4-mediated NOTCH activation

• Anti-EGFL7 effects: Treatment with an anti-EGFL7 blocking antibody:

  • Resulted in reactivation of NOTCH signaling

  • Increased differentiation and apoptosis of AML cells

  • When combined with DLL4, significantly increased NOTCH activation and induced apoptosis

• In vivo efficacy: Treatment with anti-EGFL7 alone in three different AML mouse models resulted in increased survival, supporting EGFL7 as a novel therapeutic target in AML .

What are the dose-dependent effects of anti-EGFL7 antibodies in experimental models?

Anti-EGFL7 antibodies show important dose-dependent effects that are crucial for translational research:

How does the relationship between EGFL7 and miR-126 complicate research interpretations?

The relationship between EGFL7 and microRNA-126 (miR-126) presents important considerations for researchers:

• Genomic relationship: miR-126 is encoded within intron 7 of the EGFL7 gene, meaning genetic manipulations of EGFL7 may inadvertently affect miR-126 expression .

• Knockout model considerations: Different knockout strategies affect this relationship:

  • Constitutive EGFL7 KO mice created using a retroviral gene trap vector showed ~80% reduction in miR-126 expression

  • Newer conditional models (EGFL7fl/fl;Cdh5-CreERT2) allow specific deletion of EGFL7 without affecting miR-126 expression

  • Dedicated miR-126 KO mice have been created where the miR-126 locus was replaced without affecting EGFL7

• Expression independence: In glioma specimens, EGFL7 expression in blood vessels occurred independently of miR-126/126*, allowing for separate study of their functions .

• Experimental controls: When studying EGFL7 function, researchers should include appropriate controls to distinguish EGFL7-specific effects from those potentially mediated by changes in miR-126 expression.

What are the optimal techniques for detecting EGFL7 in tissue samples?

For optimal detection of EGFL7 in tissue samples, researchers should consider:

• Antibody selection: Use validated antibodies with confirmed specificity, such as Rabbit Recombinant Monoclonal EGFL7 antibody [EPR22603-113] which is suitable for Western blotting, immunocytochemistry/immunofluorescence, and immunohistochemistry-paraffin sections .

• Positive controls: Include tissues known to express EGFL7, such as:

  • Human placenta

  • Human umbilical vein endothelial cells (HUVECs)

  • Vascular structures in brain tissue sections

• Negative controls: Include Jurkat cells (human T cell leukemia) as a negative control for EGFL7 expression .

• Colocalization studies: Combine EGFL7 staining with endothelial markers (CD31), pericyte markers (PDGFRβ), smooth muscle cell markers (SMA), or basement membrane components (Col IV) to assess vascular expression and maturation .

• Specificity validation: Perform costaining with multiple anti-EGFL7 antibodies from different sources to confirm specificity, as demonstrated in glioma specimens where different antibodies showed 100% overlap .

What are the considerations for using anti-EGFL7 antibodies in different experimental techniques?

When using anti-EGFL7 antibodies across different experimental applications, researchers should consider:

Western Blotting:

• Use 5% non-fat dry milk in TBST as blocking buffer

• Typical antibody dilution: 1/1000

• Expected molecular weight consistent with literature (PMID: 18497746)

• Include positive controls (placenta, HUVEC) and negative controls (Jurkat cells)

Immunohistochemistry:

• Paraffin sections work well for EGFL7 detection

• Pay special attention to vascular structures, as they typically show the strongest EGFL7 staining

• Large blood vessels with distinct lumen yield particularly strong EGFL7 signals

• Quantification methods should include the percentage of EGFL7-positive vessels among all intratumoral vessels

Functional Assays:

• For blocking activities of EGFL7, select antibodies that inhibit both adhesive and prosurvival activities

• Screen for antibodies that bind to both human and mouse EGFL7 with comparable affinities for translational research

• Confirm that binding affinity and in vitro activities are preserved after humanization of therapeutic antibodies

What are the potential applications of anti-EGFL7 therapy beyond cancer?

Anti-EGFL7 therapies have potential applications beyond cancer:

• Multiple sclerosis and neuroinflammation: Since EGFL7 can limit CNS immune infiltration, modulating EGFL7 expression or function represents a potential therapeutic avenue in MS. Treatment with recombinant EGFL7 improved EAE (an MS model), reduced MCAM expression, and tightened the blood-brain barrier in mice .

• Graft-versus-host disease: Treatment with recombinant EGFL7 in murine models of acute GVHD decreased disease severity and improved survival in recipient mice after allogeneic transplantation without affecting the graft-versus-leukemia effect .

• Vascular disorders: Given EGFL7's role in regulating vascular development and homeostasis, therapeutic targeting might be relevant in conditions characterized by abnormal vessel formation or function, such as diabetic retinopathy or cardiovascular diseases.

• Inflammatory conditions: EGFL7's ability to modulate endothelial cell activation and T-cell migration suggests potential applications in other inflammatory conditions where endothelial dysfunction plays a key role.

How might combination therapies with anti-EGFL7 and other agents be optimized?

Optimizing combination therapies with anti-EGFL7 antibodies requires several considerations:

What emerging technologies might advance EGFL7 research?

Several emerging technologies could advance EGFL7 research:

• Molecular imaging techniques: Transmission electron microscopy (TEM), molecular modeling, and imaging TEM with class sum image calculations have been employed to visualize EGFL7 structure and interactions . Further advancements in these techniques could provide deeper insights into EGFL7's molecular mechanisms.

• Single-cell analysis: Single-cell RNA sequencing could help identify cell type-specific EGFL7 expression patterns and responses to anti-EGFL7 therapy in heterogeneous tissues.

• Organoid models: Patient-derived organoids might provide more physiologically relevant systems to study EGFL7's effects on vascular development and tumor growth.

• CRISPR-Cas9 technology: More precise genetic manipulation of EGFL7 without affecting miR-126 could allow clearer delineation of EGFL7-specific functions.

• Biomarker development: Further refinement of circulating progenitor cells as biomarkers, possibly combined with liquid biopsy approaches, could improve monitoring of anti-EGFL7 therapy responses in clinical settings.

• Antibody engineering: Development of next-generation anti-EGFL7 antibodies with improved binding characteristics, tissue penetration, or effector functions could enhance therapeutic efficacy.