FLT1 D4 Human

What is the molecular structure of human FLT1 D4?

Human FLT1 D4 (sVEGFR-1(D4)) is a non-chimeric, monomeric protein containing only the first 4 extracellular domains of the VEGFR-1 receptor. These domains contain all necessary information for VEGF binding. The receptor monomers have a molecular weight of approximately 55 kDa and contain 457 amino acid residues. The full VEGFR-1 receptor normally contains seven immunoglobulin-like extracellular domains, a single transmembrane region, and an intracellular split tyrosine kinase domain, but the D4 variant includes only the first four Ig-like domains . This structure allows FLT1 D4 to maintain high binding affinity for VEGF without signal transduction capability.

How does FLT1 D4 differ from full-length VEGFR-1?

FLT1 D4 represents a truncated form of the full VEGFR-1 receptor, containing only domains D1-D4 of the extracellular portion. The full-length human VEGFR1 contains 1312 amino acids forming a mature ~180 kDa glycoprotein with seven immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic domain . While the full-length receptor mediates cellular signaling upon VEGF binding, FLT1 D4 functions primarily as a ligand trap. FLT1 D4 retains the high-affinity binding to VEGF-A (~2-10 picomolar affinity), VEGF-B, and PlGF, but lacks signaling capability due to the absence of transmembrane and intracellular domains . Naturally occurring soluble VEGFR-1 is generated through alternative splicing of the flt-1 mRNA and serves as an endogenous regulator of angiogenesis .

What are the binding partners of FLT1 D4?

FLT1 D4 binds to several members of the VEGF family with varying affinities:

• VEGF-A: Binds with very high affinity (~2-10 picomolar Kd), which is much higher than VEGFR2

• VEGF-B: Binds specifically to VEGFR1/FLT1 and not other VEGF receptors

• PlGF (Placental Growth Factor): Binds with an affinity of ~170 picomolar (Kd)

This selective binding profile makes FLT1 D4 an important tool for studying differential VEGF signaling pathways and developing targeted therapeutic approaches to modulate specific VEGF-dependent processes.

How can I assess the biological activity of recombinant FLT1 D4?

The biological activity of sVEGFR-1(D4) can be determined by measuring its ability to inhibit VEGF-A-induced proliferation of Human Umbilical Vein Endothelial Cells (HUVECs) . A methodological approach includes:

• Culture HUVECs in appropriate growth medium with reduced serum (0.5-2%)

• Pre-incubate various concentrations of recombinant FLT1 D4 with a fixed concentration of VEGF-A (10-20 ng/mL) for 30-60 minutes

• Add the mixture to HUVEC cultures and incubate for 24-72 hours

• Assess proliferation using standard methods (MTT/MTS assay, BrdU incorporation, or cell counting)

• Calculate IC50 values by plotting inhibition percentage against FLT1 D4 concentration

• Compare results with a reference standard of known activity

Effective FLT1 D4 will show dose-dependent inhibition of VEGF-A-stimulated proliferation, confirming its ability to sequester VEGF and prevent receptor activation.

What are the optimal reconstitution and storage conditions for FLT1 D4?

For optimal experimental results, proper handling of lyophilized FLT1 D4 is essential. Based on standard recombinant protein protocols:

• Reconstitution: Dissolve lyophilized sVEGFR-1(D4) in sterile, buffered solution (PBS, pH 7.4) to a concentration of 0.1-1.0 mg/mL

• Gentle mixing: Avoid vigorous shaking or vortexing to prevent protein denaturation

• Short-term storage: Store reconstituted protein at 4°C for up to 1 week

• Long-term storage: Prepare small aliquots and store at -20°C to -80°C

• Avoid freeze-thaw cycles: Repeated freezing and thawing can reduce protein activity

• Working solutions: Dilute in appropriate buffer containing carrier protein (0.1-0.5% BSA) to prevent adhesion to tubes and loss of activity

Following these guidelines helps maintain the structural integrity and biological activity of FLT1 D4 for experimental applications.

What cell models are most appropriate for studying FLT1 D4 function?

Several cellular models can be employed to study FLT1 D4 function, each offering distinct advantages:

• HUVECs (Human Umbilical Vein Endothelial Cells):

  • Express both VEGFR1 and VEGFR2

  • Well-established model for angiogenesis research

  • Suitable for proliferation, migration, and tube formation assays

• Monocytes/Macrophages:

  • Express VEGFR1 but minimal VEGFR2

  • Useful for studying VEGFR1-specific effects independent of VEGFR2

• Trophoblast cells:

  • Express high levels of VEGFR1

  • Appropriate for pre-eclampsia research models

• Muscular dystrophy cell models (e.g., mdx mouse-derived cells):

  • Useful for studying FLT1 D4 effects on muscle regeneration and vascularization

• Reporter cell lines:

  • Engineered cells with VEGF-responsive elements driving reporter gene expression

  • Allow quantitative measurement of VEGF sequestration by FLT1 D4

The choice of model should align with specific research questions, considering the native expression patterns of VEGF receptors and the cellular responses being studied.

How does FLT1 D4 modulate pathological angiogenesis in disease models?

FLT1 D4 exhibits context-dependent effects on pathological angiogenesis across different disease models:

• Duchenne Muscular Dystrophy (DMD):

  • Endothelial cell-specific Flt1 deletion resulted in increased vascular density in mdx mice (DMD model)

  • Improved muscle histology and function were observed

  • FLT1 inhibition using anti-FLT1 peptides or monoclonal antibodies blocking VEGF-FLT1 binding ameliorated the muscular dystrophy phenotype

  • Suggests that reducing FLT1 activity may be beneficial in DMD by promoting functional angiogenesis

• Pre-eclampsia:

  • Elevated sFlt1 (including D4 domain) is implicated in pre-eclampsia pathogenesis

  • Adenovirus-mediated sFlt-1 overexpression in mice caused pre-eclampsia-like symptoms

  • Histological changes included severe endotheliosis and occlusion of capillary lumens

  • Co-administration of VEGF adenovirus with sFlt-1 adenovirus rescued the phenotype

  • Suggests that excess sFlt1 contributes to pre-eclampsia through VEGF sequestration

• Corneal Neovascularization:

  • FLT1 is associated with corneal neovascularization

  • Modulation of FLT1 D4 levels could potentially regulate pathological vessel growth in corneal disease

These findings highlight the dual nature of FLT1 D4 in angiogenesis regulation—its inhibition may benefit certain conditions (DMD) while its overexpression contributes to others (pre-eclampsia).

What are the structural determinants of VEGF-FLT1 D4 binding affinity?

The high-affinity binding between VEGF and FLT1 D4 is determined by specific structural features:

VEGF-A binds to VEGFR1 with approximately 10-fold higher affinity (~2–10 picomolar Kd) compared to VEGFR2, despite VEGFR2 being the primary signaling receptor . This paradox is explained by:

• The Ig-like domain 2 of VEGFR1 forms a hydrophobic binding pocket that accommodates VEGF with high complementarity

• Key residues within domains 2-3 create specific hydrogen bonds and salt bridges with VEGF

• The orientation of the domains creates an optimal binding configuration

Understanding these structural determinants enables rational design of modified FLT1 D4 variants with altered binding properties for therapeutic applications.

How can FLT1 D4 be exploited for therapeutic development?

FLT1 D4's potential as a therapeutic agent or target derives from its ability to sequester VEGF family ligands with high affinity. Research-based therapeutic approaches include:

• For conditions with excessive VEGF signaling:

  • Recombinant FLT1 D4 administration as a VEGF trap

  • Gene therapy approaches to increase local FLT1 D4 production

  • Small molecules that mimic FLT1 D4 binding pocket structure

• For conditions with insufficient angiogenesis:

  • Anti-FLT1 D4 antibodies that prevent VEGF sequestration

  • FLT1 gene deletion or knockdown in specific cell types

  • Peptide antagonists that disrupt VEGF-FLT1 D4 interaction

• Disease-specific applications:

  • Duchenne Muscular Dystrophy: FLT1 inhibition using anti-FLT1 peptides or monoclonal antibodies improved muscle histology and function in mdx mice

  • Pre-eclampsia: Reducing circulating sFlt-1 levels alleviates symptoms in mouse models

  • Cancer: Modulation of tumor angiogenesis through targeted FLT1 D4 delivery

Translational development requires careful consideration of tissue specificity, timing of intervention, and potential off-target effects due to the widespread role of VEGF signaling in physiological processes.

What techniques are available for detecting and quantifying FLT1 D4 in biological samples?

Several complementary techniques can be employed to detect and quantify FLT1 D4:

• Enzyme-Linked Immunosorbent Assay (ELISA):

  • Commercial kits available for human sFLT1 detection

  • Sensitivity typically in the pg/mL range

  • Can be developed with antibodies specific to domains D1-D4

• Western Blotting:

  • Detect FLT1 D4 protein using antibodies against the extracellular domain

  • Distinguish from full-length receptor by molecular weight (~55 kDa vs. ~180 kDa)

  • Useful for semi-quantitative analysis in tissue or cell lysates

• Flow Cytometry:

  • Measure cell-bound FLT1 D4 using fluorescent-labeled antibodies

  • Assess binding to cell surface receptors or internalization

• Surface Plasmon Resonance (SPR):

  • Real-time measurement of binding kinetics between FLT1 D4 and VEGF variants

  • Determine association/dissociation constants

  • Useful for comparing engineered FLT1 D4 variants

• Mass Spectrometry:

  • Identify and quantify FLT1 D4 with high specificity

  • Distinguish between different splicing variants

  • Characterize post-translational modifications

These methods can be combined for comprehensive analysis of FLT1 D4 in experimental and clinical samples.

What animal models are optimal for studying FLT1 D4 function in vivo?

Several animal models have proven valuable for investigating FLT1 D4 functions:

• Genetic Models:

  • Constitutive Flt1 knockout mice (embryonic lethal due to vascular overgrowth)

  • Conditional Flt1 knockout mice (CAG:CreERTM Flt1LoxP/LoxP)

  • Endothelial cell-specific Flt1 deletion (Cdh5:CreERT2 Flt1LoxP/LoxP)

  • Tyrosine kinase domain-deficient Flt1 mice (normal development)

• Disease-Specific Models:

  • mdx mice (Duchenne muscular dystrophy model) crossed with Flt1 knockout mice

  • Adenovirus-mediated sFlt-1 overexpression for pre-eclampsia models

• Molecular Tool-Based Models:

  • Anti-FLT1 peptide or monoclonal antibody administration

  • Adenoviral co-expression of VEGF and sFlt-1 for rescue experiments

  • CRISPR/Cas9-engineered variants of Flt1 with specific domain modifications

When designing in vivo studies, consider:

• Temporal control of Flt1 modification (development vs. adult)

• Tissue specificity of intervention

• Physiological vs. pathological contexts

• Relevant endpoints (vascular density, function, disease parameters)

Each model offers distinct advantages for addressing specific research questions related to FLT1 D4 biology.

How can I investigate FLT1 D4 interactions with the VEGF signaling network?

Investigating FLT1 D4 within the complex VEGF signaling network requires multi-level approaches:

• Protein-Protein Interaction Analysis:

  • Co-immunoprecipitation of FLT1 D4 with VEGF family members

  • Biolayer interferometry or SPR for kinetic binding parameters

  • Proximity ligation assays to detect interactions in situ

  • Competitive binding assays with different VEGF family members

• Signaling Pathway Analysis:

  • Phosphorylation status of downstream effectors (ERK1/2, AKT, p38)

  • Gene expression profiling following FLT1 D4 modulation

  • Calcium flux measurement in response to VEGF with/without FLT1 D4

  • Real-time analysis of signaling using FRET-based biosensors

• Functional Cellular Responses:

  • Endothelial cell proliferation, migration, and tube formation assays

  • Analysis of tip/stalk cell differentiation in sprouting assays

  • Integration with other angiogenic pathways (Notch, Angiopoietin)

  • Effects on pericyte recruitment and vessel maturation

• Computational Approaches:

  • Systems biology modeling of VEGF/VEGFR interactions

  • Prediction of FLT1 D4 effects on VEGF gradient formation

  • Network analysis of transcriptional responses to FLT1 D4 modulation

These methods collectively provide a comprehensive understanding of how FLT1 D4 integrates into and modulates the broader VEGF signaling network across different cellular contexts.

How does hypoxia regulate FLT1 D4 expression and function?

Hypoxia is a key regulator of FLT1 expression through multiple mechanisms:

• Transcriptional Regulation:

  • VEGFR1 expression is upregulated by hypoxia through the HIF-1 complex

  • The VEGFR1 promoter contains a hypoxia-responsive element (HRE) sequence

  • HIF-1 binds to this sequence to stimulate transcription of the VEGFR1 locus

• Alternative Splicing:

  • Hypoxic conditions can alter the splicing patterns of FLT1 mRNA

  • This may affect the ratio of full-length receptor to soluble forms including FLT1 D4

  • Splicing factors regulated by hypoxia contribute to this process

• Methodological approaches to study hypoxic regulation:

  • Compare FLT1 D4 expression in normoxic vs. hypoxic conditions (1-5% O₂)

  • Use chemical mimetics of hypoxia (CoCl₂, DMOG, DFO)

  • Employ HIF-1α knockout or knockdown models

  • Analyze HIF-1 binding to the VEGFR1 promoter using ChIP assays

  • Investigate splicing factor activity under hypoxic conditions

Understanding hypoxic regulation of FLT1 D4 has implications for numerous pathological conditions where hypoxia and aberrant angiogenesis coincide, including cancer, ischemic diseases, and pre-eclampsia.

What are the contradictions in FLT1 D4 research data across different disease models?

Research on FLT1 D4 has revealed apparent contradictions that highlight its context-dependent roles:

• Opposing effects in different diseases:

  • In muscular dystrophy (mdx mice), inhibition of FLT1 is beneficial, improving vascular density and muscle function

  • In pre-eclampsia models, increased sFLT1 (including D4 domain) is detrimental, causing endothelial dysfunction and other symptoms

  • These contradictory findings suggest tissue-specific and context-dependent roles

• Molecular paradoxes:

  • VEGFR1/FLT1 has higher affinity for VEGF (~2-10 pM) than VEGFR2 (~100-125 pM) but weaker signaling activity

  • VEGFR1 knockout is embryonically lethal, but tyrosine kinase domain-deficient VEGFR1 mice develop normally

  • This suggests that the ligand-binding function (present in FLT1 D4) is more critical than signaling activity

• Resolving contradictions:

  • Consider tissue-specific VEGF dependency and receptor expression patterns

  • Examine temporal aspects of FLT1 D4 function during development vs. adult homeostasis

  • Investigate concentration-dependent effects (partial vs. complete inhibition)

  • Analyze compensatory mechanisms that may differ between acute intervention and genetic models

These apparent contradictions reflect the complex biology of FLT1 D4 and highlight the importance of precisely defined experimental conditions and careful interpretation of results across models.

How can single-cell analysis advance our understanding of FLT1 D4 biology?

Emerging single-cell technologies offer unprecedented insights into FLT1 D4 biology:

• Single-cell RNA sequencing (scRNA-seq):

  • Reveals heterogeneity in FLT1 expression across endothelial subtypes

  • Identifies cell populations that predominantly express soluble vs. membrane-bound forms

  • Maps correlations between FLT1 D4 expression and other angiogenic regulators

  • Captures dynamic changes during development or disease progression

• Single-cell protein analysis:

  • Mass cytometry (CyTOF) with FLT1 D4-specific antibodies

  • High-parameter flow cytometry to correlate FLT1 D4 with cellular phenotypes

  • Imaging mass cytometry for spatial context of FLT1 D4 expression

• Spatial transcriptomics:

  • Maps FLT1 D4 expression in tissue context

  • Correlates with vascular patterns and disease features

  • Reveals microenvironmental influences on FLT1 D4 expression

• Functional single-cell assays:

  • Microfluidic platforms to assess individual cell responses to FLT1 D4

  • Live-cell imaging of VEGF gradient formation with variable FLT1 D4 levels

  • Single-cell secretion analysis to quantify FLT1 D4 release