TIE1 Fc Mouse

What is TIE1 and what is the molecular structure of recombinant mouse TIE1 Fc protein?

TIE1 (tyrosine kinase with Ig and EGF homology domains 1) is a receptor tyrosine kinase (RTK) primarily expressed on endothelial and hematopoietic progenitor cells. It plays critical roles in angiogenesis, vasculogenesis, and hematopoiesis .

The recombinant mouse TIE1 Fc fusion protein consists of:

• The soluble extracellular domain of mouse TIE1 fused with the Fc part of human IgG1

• Produced in CHO cells as a monomeric, glycosylated polypeptide

• Contains 749 amino acids with a total molecular mass of approximately 260 kDa

• The TIE1 Fc monomer has a calculated molecular mass of approximately 105 kDa

• Due to glycosylation, it migrates as an approximately 130 kDa protein in SDS-PAGE under reducing conditions

The structure includes two immunoglobulin-like domains flanking three epidermal growth factor (EGF)-like domains, followed by three fibronectin type III-like repeats in the extracellular region .

How does TIE1 differ from TIE2 in terms of function and ligand binding?

While TIE1 and TIE2 are related receptor tyrosine kinases, they have distinct functions and ligand-binding properties:

TIE1:

• Has long been considered a ligand-less receptor

• Recent research has identified Svep1 as a binding ligand of TIE1

• Functions in a context-dependent manner by forming heterodimers with TIE2

• Can either block or activate TIE signaling depending on the cellular context

TIE2:

• Binds directly to angiopoietins (Ang1 and Ang2)

• Activates downstream signaling pathways upon ligand binding

• Knockout in mice leads to death at E9.5-10.5 due to defective cardiac development and vascular remodeling

This distinction is important when designing experiments, as TIE1-related phenotypes may be independent of or complementary to TIE2-mediated effects, as evidenced by the different phenotypes observed in zebrafish mutants .

How can TIE1 Fc Mouse be used to study tumor angiogenesis?

TIE1 Fc Mouse proteins are valuable tools for investigating tumor angiogenesis through several methodological approaches:

• Competitive inhibition studies: The soluble TIE1 Fc can be used to block endogenous TIE1 signaling pathways, allowing researchers to assess the functional significance of TIE1 in tumor vessel formation.

• Analysis of vessel morphology: Research has shown that TIE1 deletion affects the pattern of tumor blood vessels. When TIE1 is present, vessels grow in a branching honeycomb-like pattern, whereas TIE1 deletion results in decreased density of angiogenic sprouts and filopodial extensions .

• Endothelial cell survival assessment: TIE1 has been reported to promote endothelial cell survival in late-phase angiogenic capillary growth. Researchers can use TIE1 Fc to investigate this mechanism in tumor models by analyzing markers of endothelial apoptosis .

• Combination therapy models: TIE1 deletion combined with anti-angiogenic therapies (such as VEGF or VEGFR-2 blocking antibodies) can be studied using TIE1 Fc in conjunction with other inhibitors to evaluate potential synergistic effects on tumor growth inhibition .

What experimental evidence demonstrates TIE1's role in lymphangiogenesis?

Recent research provides compelling evidence for TIE1's role in lymphangiogenesis:

• Zebrafish model studies: In zebrafish, tie1 mutants exhibit significant lymphatic defects that are distinct from tie2 mutants:

  • Impaired formation of the facial cardinal lymphatic vessel (FCLV)

  • Absence of brain lymphatic endothelial cells (BLECs) at 3 days post-fertilization

  • Reduced numbers of parachordal lymphangioblasts (PLs)

• Mouse knockout studies: Tie1 knockout mice display:

  • Hemorrhages from E13.5 to P0 that lead to death

  • Lymphatic defects and edema formation from E12.5 onwards

  • Conditional Tie1 deletion results in impaired postnatal lymphatic capillary network development

• Comparative analysis: Studies comparing tie1 and svep1 mutants revealed that they share similar lymphatic phenotypes, suggesting a functional connection in the same pathway .

These findings collectively highlight TIE1's crucial role in lymphatic vessel development, which is mechanistically distinct from its functions in blood vessel formation.

What are the optimal storage and reconstitution protocols for TIE1 Fc Mouse proteins?

For optimal results when working with TIE1 Fc Mouse proteins, follow these methodological guidelines:

Storage:

• TIE1 Fc Chimera is typically supplied as a sterile filtered white lyophilized (freeze-dried) powder

• Store the lyophilized protein at -20°C to -80°C for long-term stability

• Avoid repeated freeze-thaw cycles of reconstituted protein

Reconstitution Protocol:

• Allow the lyophilized protein to reach room temperature

• Reconstitute in sterile 1× PBS (as the protein is lyophilized from a 1 mg/ml solution containing 1× PBS)

• Gently mix by rotating or swirling rather than vortexing to prevent protein denaturation

• Allow complete reconstitution (typically 10-20 minutes) before use

• For dilute solutions, consider adding a carrier protein (0.1% BSA) to prevent adsorption to tubes

Working Solution Preparation:

• Prepare working solutions immediately before use

• If necessary, filter through a 0.22 μm filter for cell culture applications

• Check endotoxin levels if using in cell culture (testing recommended prior to use)

How can researchers verify the activity and specificity of TIE1 Fc in experimental settings?

Verifying the activity and specificity of TIE1 Fc requires multiple complementary approaches:

Analytical Verification:

• SDS-PAGE analysis: Confirm protein integrity and molecular weight (should migrate as approximately 130 kDa protein under reducing conditions)

• Western blotting: Use anti-TIE1 and anti-Fc antibodies to verify identity

• RP-HPLC analysis: Ensure purity greater than 90%

Functional Validation:

• Binding assays: Confirm binding to recently identified ligands like Svep1

  • ELISA-based binding assays with recombinant Svep1

  • Surface plasmon resonance to measure binding kinetics

• Competition assays: Demonstrate ability to compete with native TIE1 for ligand binding

• Cell-based assays:

  • Monitor phosphorylation status of downstream signaling molecules

  • Assess endothelial cell survival in the presence of TIE1 Fc

Specificity Controls:

• Use an irrelevant Fc-fusion protein as negative control

• Include TIE2 Fc to distinguish TIE1-specific from TIE2-specific effects

• Validate with genetic models (e.g., compare effects with Tie1 knockout phenotypes)

How should researchers address contradictory results between in vitro TIE1 Fc studies and in vivo genetic models?

When facing contradictory results between TIE1 Fc in vitro studies and in vivo genetic models, consider these methodological approaches:

• Evaluate model-specific differences:

  • Acute inhibition (TIE1 Fc) versus chronic depletion (genetic knockout)

  • Developmental timing (embryonic versus adult tissues)

  • Species-specific differences (e.g., zebrafish versus mouse models)

• Assess receptor heterodimerization effects:

  • TIE1 can form heterodimers with TIE2, affecting angiopoietin signaling

  • In vitro systems may not fully recapitulate the complex interaction patterns present in vivo

  • Examine TIE1-TIE2 heterodimer formation in your experimental system

• Analyze context-dependent signaling:

  • The tumor microenvironment differs substantially from normal tissues

  • In normal vasculature, TIE1 deletion may not affect vessel density, while tumor vessels show significant changes

  • Compare results across multiple tissue contexts

• Experimental validation approaches:

  • Rescue experiments using TIE1 Fc in Tie1-deficient models

  • Cross-validation with multiple genetic models (conditional knockouts, hypomorphs)

  • Combined in vitro/in vivo approaches to bridge the gap between systems

What are the potential confounding factors when studying TIE1 Fc effects on tumor angiogenesis?

When studying TIE1 Fc effects on tumor angiogenesis, researchers should consider these potential confounding factors:

• Inflammatory responses:

  • TIE1 has been suggested to play a role in inflammation

  • Changes in endothelial expression of leukocyte adhesion receptors (P-selectin, ICAM1) may affect vascular function

  • Carefully assess inflammatory cell recruitment (CD45+, F4/80+, Lys6/Gr-1+ cells) to distinguish direct TIE1 effects from inflammatory-mediated effects

• Compensatory mechanisms:

  • Upregulation of alternative angiogenic pathways (e.g., VEGF) may mask TIE1 inhibition effects

  • Time-dependent adaptations may occur in long-term studies

  • Consider analyzing multiple timepoints and combination treatments

• Tumor model variability:

  • Different tumor models (LLC, B16F10, EL4) may show varying dependencies on TIE1 signaling

  • Tumor cell-specific effects should be distinguished from host endothelial effects

  • Use multiple tumor models to validate findings

• Glycosylation heterogeneity:

  • TIE1 Fc is a glycosylated protein with molecular weight variability

  • Production systems (e.g., CHO cells) may introduce glycosylation patterns not present in vivo

  • Batch-to-batch variations in glycosylation may affect activity

How does the newly identified Svep1-TIE1 interaction impact our understanding of vascular development?

The identification of Svep1 as a TIE1 ligand represents a significant breakthrough that reshapes our understanding of vascular development:

• Paradigm shift in TIE1 biology:

  • TIE1 was long considered an orphan receptor without identified ligands

  • The discovery of Svep1 as a binding partner provides a mechanistic explanation for TIE1's functions independent of TIE2

  • This finding suggests that TIE1 has direct signaling capabilities rather than solely modulating TIE2 activity

• Specific developmental roles:

  • Svep1-TIE1 interaction appears crucial for specific aspects of lymphatic development:

    • Formation of the facial cardinal lymphatic vessel (FCLV)

    • Development of brain lymphatic endothelial cells (BLECs)

    • Proper parachordal lymphangioblast (PL) formation

  • These processes occur independently of VEGFC and TIE2 signaling

• Evolutionary conservation:

  • Both zebrafish and human SVEP1 proteins bind to their respective TIE1 receptors in vitro

  • This conservation suggests fundamental importance in vertebrate vascular development

• Clinical relevance:

  • Compound heterozygous mutations for SVEP1 and TIE2 have been reported in human glaucoma patients

  • This genetic interaction demonstrates clinical significance of SVEP1 in TIE signaling

What are the implications of combining TIE1 targeting with other angiogenesis inhibitors?

Research on combining TIE1 targeting with other angiogenesis inhibitors reveals important considerations for developing more effective anti-angiogenic therapies:

Table 1: Comparison of TIE1, TIE2, and SVEP1 Knockout Phenotypes in Different Model Systems

Table 2: Properties of Recombinant Mouse TIE1 Fc Protein