Endoglin (27-581) Mouse

What is Endoglin (27-581) Mouse and what is its structure?

Endoglin (27-581) Mouse is a recombinant protein comprising amino acids 27-581 of mouse endoglin, produced in Sf9 Baculovirus cells. It is a single, glycosylated polypeptide chain containing 563 amino acids with a molecular mass of 60.9 kDa, though it typically migrates at 50-70 kDa on SDS-PAGE under reducing conditions due to glycosylation . The protein is expressed with an 8-amino acid His tag at the C-terminus to facilitate purification through chromatographic techniques .

Native endoglin exists as a homodimer of approximately 180 kDa with disulfide linkages, and this recombinant form retains the key extracellular domains while excluding the transmembrane and cytoplasmic portions found in the full-length protein . The amino acid sequence includes several conserved domains that are critical for interaction with TGF-β family ligands.

What is the role of Endoglin in TGF-β signaling pathways?

Endoglin functions as an accessory receptor for the TGF-β receptor complex, modulating signaling responses to various TGF-β family ligands. It is particularly important for regulating the specific binding of TGF-β1 to its receptors and has been found to be part of the TGF-β1 receptor complex . Recent research has demonstrated that endoglin specifically binds to BMP9 and BMP10, with these interaction domains being highly conserved between human and mouse species .

This binding specificity allows endoglin to influence multiple downstream signaling pathways, including both Smad1/5 (via BMP signaling) and Smad2/3 (via TGF-β signaling). The balance between these pathways is critical for regulating various cellular processes including proliferation, differentiation, migration, and survival, particularly in endothelial cells where endoglin is predominantly expressed.

Where is endoglin expressed and what does this suggest about its function?

Endoglin is predominantly expressed on endothelial cells, especially those undergoing active angiogenesis. Additionally, it has been found on activated macrophages, fibroblasts, and smooth muscle cells . This expression pattern points to endoglin's multifaceted roles in vascular biology and tissue homeostasis.

The high expression on endothelial cells suggests critical functions in vascular development, maintenance, and remodeling. Its presence on activated macrophages indicates involvement in immune responses and inflammation. Expression on fibroblasts and smooth muscle cells further suggests roles in tissue repair, fibrosis, and regulation of vascular tone.

This diverse expression profile explains why dysregulation of endoglin is associated with various pathological conditions related to the cardiovascular system, including hereditary hemorrhagic telangiectasia, preeclampsia, and certain cardiovascular disorders .

How does soluble endoglin differ from membrane-bound endoglin?

Soluble endoglin (sEng) represents the extracellular domain of endoglin that has been cleaved from the cell surface. While membrane-bound endoglin is anchored to cell membranes and can participate in intracellular signaling through its cytoplasmic domain, soluble endoglin circulates freely and lacks these signaling capabilities.

This distinction is crucial for understanding the different roles these forms play in physiological and pathological processes. While membrane-bound endoglin facilitates signaling, soluble endoglin may act as a decoy receptor, potentially neutralizing TGF-β1 signaling by inhibiting binding to the TGF-β receptor complex .

How does Endoglin (27-581) Mouse specifically interact with BMP9 and BMP10?

Recent research has demonstrated that mouse endoglin constructs (including the 27-581 region) specifically bind to human BMP9 and BMP10 . This interaction is particularly significant because it indicates that the binding domains between endoglin and these bone morphogenetic proteins are highly conserved across species.

The interaction between endoglin and BMP9/10 has important implications for vascular biology research. When endoglin binds to these growth factors, it can modulate their interaction with type I and type II receptors, consequently affecting the Smad1/5 signaling pathway . This modulation capability makes endoglin a critical determinant of signaling balance and specificity in endothelial cells.

What effects does soluble endoglin have on endothelial function in vivo?

Studies using transgenic mice expressing high levels of human soluble endoglin (Sol-Eng+) have provided valuable insights into how soluble forms of endoglin affect endothelial function. These transgenic mice exhibit higher plasma concentrations of human soluble endoglin (2,000-3,000 ng/mL) compared to control mice, and they develop increased blood arterial pressure .

Interestingly, despite the elevated blood pressure, functional analysis both in vivo and ex vivo in isolated aorta demonstrated that endothelium-dependent vascular function remained similar in Sol-Eng+ and control mice . Western blot analysis showed no differences between these mice in the protein expression levels of endoglin, endothelial NO-synthase (eNOS), or pro-inflammatory adhesion molecules ICAM-1 and VCAM-1 in the aorta .

Gender-specific effects have been observed, with female Sol-Eng+ mice showing impaired vascular responses to phenylephrine-induced contraction that were not seen in male mice . This suggests that soluble endoglin may interact with gender-specific hormonal factors to influence vascular function through mechanisms independent of endothelial dysfunction.

What are the methodological differences between studies using recombinant versus endogenous endoglin?

Using recombinant Endoglin (27-581) versus studying endogenous endoglin presents several important methodological considerations that can significantly impact experimental outcomes:

Localization and Distribution: Recombinant Endoglin (27-581) is soluble and freely diffuses in experimental systems, whereas endogenous endoglin is primarily membrane-bound. This fundamental difference affects protein localization, concentration gradients, and interaction kinetics with binding partners.

Signaling Capacity: Endogenous membrane-bound endoglin participates in signal transduction through its cytoplasmic domain, which is absent in the recombinant 27-581 fragment. The recombinant form may therefore act as a competitive inhibitor by binding ligands without initiating downstream signaling.

Glycosylation Patterns: Recombinant proteins produced in insect cells (like the Sf9 system used for Endoglin 27-581) may have different glycosylation patterns compared to mammalian-expressed endogenous endoglin . These differences can affect protein folding, stability, and binding affinity.

Concentration Effects: Experiments using recombinant protein often employ concentrations that may exceed physiological levels, potentially leading to non-physiological binding events or signaling outcomes. The sol-Eng+ transgenic mice, for example, have plasma concentrations of human soluble endoglin (2,000-3,000 ng/mL) that are approximately 1000 times higher than those observed in mice with advanced atherosclerosis (2,000-3,000 pg/mL) .

These differences should be carefully considered when designing experiments and interpreting results, particularly when attempting to extrapolate findings from recombinant protein studies to in vivo situations.

How can gender differences impact endoglin research outcomes?

Gender differences significantly impact experimental outcomes in endoglin studies, as demonstrated by research showing differential vascular responses in male versus female Sol-Eng+ mice . These sex-specific effects have important implications for research design and interpretation.

Studies have shown that female Sol-Eng+ mice exhibit impaired vascular responses to phenylephrine-induced contraction that are not observed in male Sol-Eng+ mice . This suggests gender-specific differences in adrenergic receptor signaling or smooth muscle cell function in response to high soluble endoglin levels. The mechanism appears to be independent of endothelial dysfunction, as endothelium-dependent vasodilation and endothelial markers remained similar between male and female mice .

These findings highlight the importance of including both male and female subjects in endoglin research and analyzing data by sex before pooling. Researchers should consider how hormonal factors might interact with endoglin signaling, potentially through controlling the study phase of estrous cycle in female animals or through hormone replacement studies.

Understanding these gender differences is crucial for developing a comprehensive understanding of endoglin biology and for the appropriate design of therapeutic strategies targeting endoglin-related pathways, especially for conditions with gender-specific prevalence or manifestations.

What are the limitations of using Endoglin (27-581) Mouse in atherosclerosis research?

Using Endoglin (27-581) Mouse in atherosclerosis research presents several significant limitations that researchers should consider:

Lack of Appropriate Transgenic Models: As noted in the literature, transgenic mice with high levels of soluble endoglin on atherosclerotic backgrounds (ApoE-deficient or LDLR-deficient) are not readily available . This limits the ability to directly assess how high levels of soluble endoglin might contribute to endothelial dysfunction in the context of hypercholesterolemia and chronic inflammation.

Concentration Discrepancies: The concentration of soluble endoglin in transgenic models (2,000-3,000 ng/mL) is approximately 1000 times higher than levels observed in hypercholesterolemic mice with advanced atherosclerosis (2,000-3,000 pg/mL) . This raises questions about the physiological relevance of findings from these models to actual atherosclerotic disease.

These limitations highlight the need for developing more sophisticated models combining soluble endoglin expression with atherosclerotic backgrounds to better understand its role in this complex disease process.

What are optimal storage and handling conditions for Endoglin (27-581) Mouse?

Proper storage and handling of Endoglin (27-581) Mouse recombinant protein is essential for maintaining its structural integrity and functional activity. Based on manufacturer recommendations:

Short-term Storage: Store at 4°C if the entire vial will be used within 2-4 weeks . The protein is typically supplied in a buffer containing phosphate-buffered saline (pH 7.4) with 10% glycerol, which helps maintain stability at this temperature .

Long-term Storage: For periods longer than 2-4 weeks, store frozen at -20°C . For extended storage periods, the addition of a carrier protein (0.1% HSA or BSA) is recommended to prevent adhesion to surfaces and maintain stability .

Freeze-Thaw Cycles: Minimize repeated freeze-thaw cycles, as these can lead to protein denaturation and loss of activity . Aliquot the protein solution into smaller volumes before freezing to avoid repeated thawing of the entire stock.

Working Solutions: When preparing working dilutions, use buffers recommended for your specific application. For cell culture applications, ensure sterility by filtering through a 0.22 μm filter.

Handling Precautions: Avoid vigorous shaking or vortexing, which can cause protein denaturation. Use low-protein-binding tubes and pipette tips to minimize loss through adsorption.

Following these guidelines will help ensure the reliability and reproducibility of experiments using Endoglin (27-581) Mouse recombinant protein.

How can researchers validate the functional activity of Endoglin (27-581) Mouse?

Validating the functional activity of Endoglin (27-581) Mouse is essential for ensuring reliable experimental results. Several complementary techniques should be employed:

Ligand Binding Assays:

• Surface Plasmon Resonance (SPR) to measure binding kinetics with known ligands like BMP9, BMP10, or TGF-β1

• Enzyme-Linked Immunosorbent Assay (ELISA) to detect specific binding to immobilized ligands

• Pull-down assays with tagged ligands followed by western blot detection

Cell-Based Functional Assays:

• Reporter gene assays using cells transfected with BMP or TGF-β responsive elements

• Phosphorylation assays for downstream signaling molecules (Smad1/5/8 for BMP signaling; Smad2/3 for TGF-β signaling)

• Endothelial cell tube formation assays to assess anti-angiogenic properties

Structural Integrity Assessments:

• SDS-PAGE under reducing conditions to confirm the expected migration pattern (50-70 kDa)

• Size-Exclusion Chromatography (SEC) to verify oligomeric state and detect aggregation

• Western blot with anti-endoglin or anti-His tag antibodies to confirm identity

Competitive Inhibition Studies:

• Demonstrate that Endoglin (27-581) Mouse can compete with membrane-bound endoglin for ligand binding

• Show dose-dependent inhibition of known endoglin-mediated cellular responses

By employing multiple validation approaches, researchers can gain confidence in the functional integrity of their Endoglin (27-581) Mouse preparations and generate more reliable experimental data.

What experimental designs are optimal for studying endoglin's role in vascular function?

Optimal experimental designs for studying endoglin's role in vascular function should incorporate multiple complementary approaches:

In Vivo Functional Assessments:

• Telemetry for continuous blood pressure monitoring, as demonstrated in studies with Sol-Eng+ mice

• Tail-cuff measurements for blood pressure in larger cohorts, which have successfully detected increased systolic pressure in Sol-Eng+ mice

• In vivo vasoactive drug responses (such as acetylcholine for endothelium-dependent vasodilation and sodium nitroprusside for endothelium-independent vasodilation)

• NOS inhibition with L-NAME to assess NO contribution to vascular tone

Ex Vivo Vascular Function Assays:

• Wire myography for isolated vessel segments to measure endothelium-dependent and -independent relaxation

• Pre-contraction with agents like phenylephrine (PHE) or prostaglandin F2α (PGF2α) followed by relaxation with acetylcholine

• Comparison of responses in the presence and absence of L-NAME to isolate NO-dependent mechanisms

Molecular Analyses:

• Western blot analysis of endothelial markers (eNOS, endoglin) and inflammatory markers (ICAM-1, VCAM-1)

• Assessment of urinary nitrite excretion as a measurement of whole-body NO production

• Signaling pathway analysis focusing on Smad phosphorylation

Experimental Considerations:

• Include both male and female animals to account for gender-specific differences

• Consider combining models (e.g., soluble endoglin expression with atherosclerotic backgrounds) for more complex disease modeling

• Use appropriate controls (e.g., transgenic littermates that do not develop high levels of soluble endoglin)

This multifaceted approach allows for comprehensive assessment of endoglin's effects on vascular function from the molecular to the whole-organism level.

What techniques can differentiate between membrane-bound and soluble endoglin?

Distinguishing between membrane-bound and soluble endoglin is crucial for understanding their respective roles. Several methodological approaches can be employed:

Biochemical Fractionation:

• Separate membrane and soluble fractions through ultracentrifugation before performing western blot analysis

• Use appropriate membrane markers (e.g., Na+/K+ ATPase) as controls to confirm proper fractionation

Antibody Selection:

• Utilize domain-specific antibodies that can differentiate between forms

• Antibodies targeting the cytoplasmic domain will recognize only the membrane-bound form

• Antibodies targeting the extracellular domain will detect both forms

Quantitative Assays:

• ELISA assays specific for soluble endoglin can quantify levels in plasma, as demonstrated in studies measuring human soluble endoglin concentrations in plasma from transgenic mice

• Comparison of soluble endoglin levels between experimental groups and controls can provide valuable insights, as seen in the significant differences between Sol-Eng+ mice and control animals

Functional Studies:

• Compare effects of recombinant soluble endoglin (like Endoglin 27-581) with those of membrane-bound endoglin

• Assess differences in signaling pathway activation (particularly Smad1/5 vs. Smad2/3 pathways)

By combining these approaches, researchers can comprehensively characterize the distribution and relative abundance of both endoglin forms in their experimental systems.

What considerations are important when designing signaling studies with Endoglin (27-581)?

When designing signaling studies with Endoglin (27-581) Mouse, researchers should consider several important factors:

Concentration Optimization:

• Determine appropriate concentration ranges through dose-response experiments

• Consider that physiological concentrations of soluble endoglin in mice with advanced atherosclerosis are around 2,000-3,000 pg/mL, while transgenic models express much higher levels (2,000-3,000 ng/mL)

• Be aware that non-physiological concentrations may lead to off-target effects

Pathway Specificity:

• Focus on both canonical TGF-β/Smad pathways and potential alternative signaling mechanisms

• Consider that endoglin modulates both Smad1/5 (via BMP signaling) and Smad2/3 (via TGF-β signaling) pathways

• Include readouts for endothelial function markers (eNOS expression and activity) and inflammatory markers (ICAM-1, VCAM-1)

Experimental Controls:

• Include both positive controls (direct ligand stimulation) and negative controls (heat-inactivated protein)

• Consider pre-incubation experiments to determine whether Endoglin (27-581) affects ligand binding to receptors

• Use appropriate inhibitors (e.g., L-NAME for NOS inhibition) to dissect specific pathway contributions

Cell Type Considerations:

• Results may vary significantly between cell types based on their endogenous expression of endoglin and TGF-β receptors

• Endothelial cells are particularly relevant given endoglin's high expression in this cell type

• Consider using primary cells where possible, as immortalized cell lines may have altered signaling properties

By systematically addressing these considerations, researchers can generate more reliable and physiologically relevant insights into endoglin's role in signaling pathways.