FLT1 D7 Mouse

What is the FLT1 D7 Mouse chimera protein and how does it function in vascular biology?

The Mouse Flt-1/VEGFR-1 (D7)-FC Chimera is a recombinant soluble protein where the vascular endothelial growth factor receptor-1 (sVEGFR-1D1-7) is fused with the Fc part of human IgG1. This creates a disulfide-linked homodimeric protein with monomers of approximately 130 kDa. The soluble receptor contains all seven extracellular domains (Met1-Thr751) necessary for high-affinity ligand binding. Functionally, this chimeric protein acts as a potent VEGF antagonist by sequestering VEGF and preventing its interaction with membrane-bound receptors . The biological activity of sVEGFR-1/Fc can be measured through its ability to inhibit VEGF-dependent proliferation of human umbilical vein endothelial cells .

How does Flt1 regulate angiogenesis at the molecular level?

Flt1 (VEGFR-1) functions primarily as a negative regulator of VEGF-A signaling by acting as a "decoy" receptor or ligand sink during vascular development . It binds VEGF-A with approximately 10-fold higher affinity than Flk1/Kdr (VEGFR-2), effectively regulating available VEGF-A ligand . The tyrosine kinase domain of Flt1 is dispensable for normal vascular development, further supporting its function as a ligand trap rather than a signaling receptor . When Flt1 is genetically deleted, excessive and aberrant Flk1 activation occurs via increased receptor phosphorylation, disrupting normal vascular patterning and leading to overgrown, poorly branched vessels .

What transgenic mouse models are available for studying Flt1 function in vivo?

Several transgenic mouse models have been developed to study Flt1 function:

The PGFD mouse model is particularly valuable as it allows for the simultaneous in vivo imaging of hemangiogenesis and lymphangiogenesis throughout experimental time courses .

How should researchers design experiments to study the effects of Flt1 modulation on blood vessel formation?

When designing experiments to study Flt1 modulation effects on angiogenesis, researchers should consider:

• Model selection: In vitro models using ESC-derived vessels or ex vivo models like aortic ring assays provide controlled environments, while in vivo models such as the corneal angiogenesis assay or retinal vascularization in PGFD mice offer physiologically relevant contexts .

• Genetic approaches: Compare wild-type (WT) with Flt1−/− models to understand complete loss-of-function effects . For tissue-specific or temporal control, use conditional knockout systems like the CDh5CreERT2 crossed with VEGFR2lox and PGFD mice with tamoxifen induction .

• Visualization methods: For Flt1-DsRed or PGFD mouse models, confocal or two-photon microscopy enables direct observation of vessel formation, branching, and regression in real-time without additional staining .

• Key endpoints: Measure vessel density, branching complexity, pericyte coverage (using markers like NG2/Cspg4), endothelial proliferation rates, and vessel permeability .

• Molecular readouts: Include analysis of downstream signaling pathways like Notch (Dll4, Hes1, Hey1, Hey2) and PDGF-B that are disrupted by Flt1 loss .

What methods are most effective for isolating and analyzing Flt1-expressing cell populations from mouse tissues?

For optimal isolation and analysis of Flt1-expressing cell populations:

• Cell isolation: Magnetic-activated cell sorting (MACS) can effectively separate endothelial cells from other cell types in differentiated ESC cultures or dissociated tissues . For greater purity, fluorescence-activated cell sorting (FACS) can isolate Flt1-DsRed-expressing cells from transgenic mouse tissues.

• RNA analysis: Quantitative RT-PCR can determine Flt1 expression levels relative to endothelial markers (Pecam1) and compare with other VEGF receptors (Flk1/Kdr) . When analyzing rare populations, consider RNA amplification methods.

• Protein detection: Western blotting for total Flt1 and phosphorylated Flt1 provides information on protein levels and activation status. Immunoprecipitation may be necessary for detecting low-abundance signaling complexes.

• Imaging approach: For transgenic Flt1-DsRed mice, direct fluorescence microscopy visualizes Flt1-expressing cells in situ . For wildtype tissues, immunohistochemistry with anti-Flt1 antibodies is required.

• Single-cell analysis: Single-cell RNA sequencing helps identify heterogeneity within Flt1-expressing populations and reveals distinct endothelial subpopulations (tip cells vs. stalk cells).

How can the soluble Mouse Flt-1/VEGFR-1 (D7)-FC Chimera be optimally reconstituted and utilized in experimental settings?

For optimal reconstitution and experimental use of soluble Mouse Flt-1/VEGFR-1 (D7)-FC Chimera:

• Reconstitution protocol: The lyophilized sVEGFR-1/Fc should be reconstituted in PBS or appropriate medium to a concentration not lower than a specific minimum threshold (typically in the µg/ml range) . Complete solubilization is critical before experimental use.

• Storage conditions: After reconstitution, the protein solution should be aliquoted to avoid repeated freeze-thaw cycles and stored at -20°C or -80°C depending on planned duration of storage.

• Functional validation: Before experimental use, validate activity through inhibition of VEGF-dependent endothelial cell proliferation assays .

• Application methods:

  • For in vitro studies: Add directly to culture medium at concentrations determined by dose-response experiments

  • For ex vivo models: Incorporate into matrigel or collagen matrices used in sprouting assays

  • For in vivo studies: Administer via local injection, osmotic pumps, or encapsulated in slow-release polymers

• Controls: Include appropriate controls such as heat-inactivated protein or irrelevant Fc-fusion proteins of similar size to validate specificity of observed effects.

How should researchers interpret conflicting Flt1 expression data between different vascular beds or developmental stages?

When confronted with conflicting Flt1 expression data:

• Contextual factors: Consider that Flt1 expression may be stage or model specific . Developmental timing, tissue microenvironment, and pathological states significantly influence expression patterns.

• Cell type specification: While endothelial cells consistently express high levels of Flt1, expression in pericytes and other perivascular cells varies considerably across studies . Analysis of purified cell populations is essential for accurate interpretation.

• Isoform consideration: Distinguish between membrane-bound Flt1 and soluble Flt1 (sFlt1), as their ratios vary across tissues and developmental stages. qRT-PCR primers should be designed to differentiate these isoforms.

• Methodological differences: Evaluate whether conflicting data arise from methodological variations (antibody specificity, sample preparation, detection sensitivity) rather than true biological differences.

• Reconciliation approach: Create a comprehensive model incorporating temporal and spatial regulation of Flt1 expression. For example, while developing retinal vessels show strong endothelial Flt1 expression, pericytes may express Flt1 only under specific conditions or in mature vessels .

What are the implications of altered Notch and PDGF-B pathway gene expression in Flt1−/− models?

The implications of altered Notch and PDGF-B pathway gene expression in Flt1−/− models include:

• Disrupted tip/stalk cell dynamics: Increased expression of Dll4, Hes1, Hey1, and Hey2 in Flt1−/− endothelial cells indicates hyperactivation of the Notch pathway . This disrupts the balanced selection of tip and stalk cells required for coordinated vessel sprouting.

• Pericyte recruitment defects: Decreased expression of Jagged1 (Jag1) in Flt1−/− endothelial cells suggests impaired endothelial-mural cell communication . This, combined with increased but potentially disorganized PDGF-B expression, explains the observed reduction in pericyte coverage despite elevated PDGF-B levels.

• ECM organization abnormalities: Increased expression of Perlecan (Hspg2) in Flt1−/− endothelial cells likely alters the distribution and retention of PDGF-B in the extracellular matrix , affecting the directional cues for pericyte migration.

• Causal relationships: The data suggest a sequence where excess VEGF-A signaling (due to Flt1 loss) first disrupts endothelial Notch signaling, which then alters PDGF-B pathway regulation, ultimately leading to impaired pericyte-endothelial interactions indirectly .

What considerations are important when quantifying blood and lymphatic vessel parameters in the PGFD mouse model?

When quantifying vascular parameters in PGFD mice:

• Signal specificity validation: Confirm the specificity of GFP and DsRed signals through co-localization with established lymphatic and blood vessel markers to exclude potential non-vascular expression .

• Imaging depth standardization: For tissues with significant thickness, standardize the imaging depth or use 3D reconstruction to avoid sampling bias, as vessel density varies across tissue depths.

• Quantification metrics:

• Dynamic vs. static measurements: For live imaging studies, develop standardized protocols for tracking vessel formation, regression, and remodeling over time rather than relying solely on endpoint analyses .

• Statistical considerations: Account for inherent variability between different anatomical regions, even within the same organ, by increasing sample sizes and using appropriate statistical methods for nested data.

How can the PGFD mouse model be utilized to investigate the interdependence of hemangiogenesis and lymphangiogenesis?

The PGFD mouse model offers unique opportunities for investigating hemangiogenesis-lymphangiogenesis interdependence:

• Simultaneous visualization: The inherent dual fluorescence allows direct visualization of both vessel types in the same tissue without additional labeling, enabling precise temporal and spatial correlation of developmental events .

• Injury models: In corneal injury or inflammation models, researchers can track how the initial blood vessel formation influences subsequent lymphatic vessel development, and whether lymphatic vessels follow similar or distinct trajectories .

• Growth factor manipulation: Implantation of pellets containing specific factors (VEGF-A, VEGF-C, etc.) enables the study of differential responses of blood versus lymphatic vessels to individual growth factors .

• Genetic modifications: Crossing PGFD mice with conditional knockout lines (e.g., CDh5CreERT2/VEGFR2lox) permits the study of how receptor deletion in one vessel type affects the other vessel system .

• Disease models: In cancer or inflammation models, PGFD mice allow tracking of both angiogenesis and lymphangiogenesis during disease progression, revealing potential therapeutic windows where targeting one process minimally impacts the other.

What molecular mechanisms explain the disruption of pericyte recruitment in Flt1−/− vessel formation despite increased PDGF-B expression?

The paradoxical disruption of pericyte recruitment despite increased PDGF-B expression in Flt1−/− vessels can be explained by:

How can researchers design experiments to distinguish between cell-autonomous and non-cell-autonomous effects of Flt1 deletion?

To distinguish between cell-autonomous and non-cell-autonomous effects of Flt1 deletion:

• Cell-specific conditional knockout systems: Utilize endothelial-specific (CDh5CreERT2) or pericyte-specific (NG2-CreER) drivers crossed with Flt1-floxed mice to delete Flt1 in specific cell populations . Compare the resulting phenotypes to distinguish direct from indirect effects.

• Chimeric approaches: Generate mosaic vessels containing both wild-type and Flt1−/− endothelial cells through:

  • Ex vivo: Co-culture of differentiating wild-type and Flt1−/− ESCs at various ratios

  • In vivo: Blastocyst complementation or bone marrow transplantation to create chimeric animals

• Cell-selective rescue experiments: In global Flt1−/− backgrounds, restore Flt1 expression specifically in endothelial cells or pericytes using cell-type-specific promoters, then assess which vascular phenotypes are rescued.

• Co-culture systems: Develop in vitro systems where:

  • Wild-type endothelial cells are cultured with Flt1−/− pericytes

  • Flt1−/− endothelial cells are cultured with wild-type pericytes

  • Compare recruitment, coverage, and gene expression patterns in each scenario

• Conditioned medium experiments: Collect conditioned medium from wild-type or Flt1−/− endothelial cells and assess its effects on pericyte migration, proliferation, and differentiation to identify secreted mediators of non-cell-autonomous effects.

What imaging techniques are most appropriate for analyzing FLT1 expression and function in different experimental contexts?

Different experimental contexts require tailored imaging approaches:

• In vivo longitudinal studies: For PGFD mice, multiphoton microscopy offers superior depth penetration with reduced phototoxicity, enabling repeated imaging of the same vessel structures over days to weeks . Imaging windows implanted over organs of interest (brain, mammary gland) further facilitate longitudinal observations.

• High-resolution cellular dynamics: Spinning disk confocal microscopy provides the temporal resolution needed to capture rapid cellular events like filopodia extension in tip cells or pericyte migration along nascent vessels in ex vivo explants.

• Molecular interaction analysis: Förster resonance energy transfer (FRET) microscopy can detect direct interactions between fluorescently tagged VEGF-A and Flt1 or between Flt1 and co-receptors in living cells, providing spatial information about signaling complexes.

• Whole-organ vascular mapping: Light sheet fluorescence microscopy combined with tissue clearing techniques (CLARITY, iDISCO) allows complete 3D reconstruction of entire vascular beds in organs from PGFD mice .

• Correlative approaches: Combining intravital fluorescence imaging of Flt1-DsRed vessels with subsequent electron microscopy of the same tissue regions provides both functional and ultrastructural information about Flt1-expressing vessels.

How should researchers approach experimental design when studying the effects of pharmacological modulators of the VEGF-Flt1 pathway?

When designing experiments with pharmacological modulators of the VEGF-Flt1 pathway:

• Compound selection strategy:

  • For Flt1 inhibition: Consider selective tyrosine kinase inhibitors, neutralizing antibodies against Flt1, or sFlt1 protein traps

  • For Flt1 activation: Consider Flt1-specific ligands (PlGF) that don't activate Flk1/VEGFR2

• Dosing considerations:

  • Establish complete dose-response curves rather than single concentrations

  • Determine both EC50 (half-maximal effective concentration) and IC50 (half-maximal inhibitory concentration) values

  • Measure actual drug concentrations in target tissues when possible

• Control experiments:

  • Include both positive controls (known modulators) and negative controls (inactive analogs)

  • Compare effects with genetic models (Flt1−/− or Flt1 overexpression) to validate specificity

• Timing considerations:

  • Test effects during different phases of vessel development (initiation, sprouting, maturation)

  • Consider acute versus chronic administration protocols

• Readout selection:

  • Primary readouts: Vessel density, branching, pericyte coverage

  • Secondary readouts: Gene expression changes in Notch and PDGF-B pathways

  • Functional readouts: Vessel permeability, blood flow, tissue perfusion

What are the most informative experimental approaches for studying the role of soluble versus membrane-bound Flt1 isoforms?

To differentiate the roles of soluble versus membrane-bound Flt1 isoforms:

• Isoform-specific genetic models:

  • Utilize mice with selective deletion of soluble Flt1 while maintaining membrane-bound expression

  • Compare with models expressing only the soluble form without the transmembrane domain

• Domain-specific functional analysis:

  • Use recombinant proteins with specific domain deletions (e.g., Mouse Flt-1/VEGFR-1 (D7)-FC vs. truncated versions)

  • Assess binding affinities, signaling capabilities, and functional outcomes of each construct

• Localized delivery approaches:

  • Matrix-bound presentation of soluble Flt1 to mimic ECM sequestration

  • Cell-surface tethering of soluble Flt1 to simulate membrane localization without signaling capacity

• Quantitative distribution analysis:

  • Develop techniques to measure the ratio of soluble to membrane-bound Flt1 in different tissues

  • Correlate this ratio with vascular phenotypes to establish physiological relevance

• Differential gene expression analysis:

  • Compare transcriptional responses to selective inhibition of each isoform

  • Identify unique downstream targets that distinguish their biological functions

How might integrating FLT1 research with emerging technologies advance vascular biology understanding?

Integration of FLT1 research with emerging technologies offers several promising directions:

• Single-cell multi-omics: Combining single-cell RNA sequencing with proteomics and metabolomics in Flt1-expressing cells could reveal previously unrecognized heterogeneity in receptor expression, signaling, and cellular responses across different vascular beds.

• CRISPR-based screens: Genome-wide or targeted CRISPR screens in endothelial cells could identify novel regulators of Flt1 expression and function, potentially revealing therapeutic targets for modulating VEGF signaling in disease.

• Organ-on-chip models: Microfluidic devices incorporating endothelial cells, pericytes, and parenchymal cells could enable high-throughput testing of how Flt1 modulation affects vascular function under controlled flow conditions and tissue-specific microenvironments.

• Computational modeling: Developing mathematical models of VEGF ligand distribution and receptor occupation that incorporate the dynamics of soluble and membrane-bound Flt1 could predict optimal intervention points for therapeutic development.

• In vivo biosensors: Developing fluorescent or luminescent biosensors that report on Flt1 activation status in real-time within living animals would transform our understanding of receptor dynamics during developmental and pathological angiogenesis.

What are the implications of FLT1 research findings for developing targeted therapies in vascular disorders?

The implications of FLT1 research for therapeutic development include:

• Isoform-specific targeting strategies: Based on the distinct roles of soluble versus membrane-bound Flt1, therapies could be designed to selectively modulate one isoform while preserving the function of the other, potentially reducing side effects.

• Pericyte-endothelial crosstalk modulation: Understanding how Flt1 affects pericyte recruitment through Notch and PDGF-B pathways suggests combination approaches targeting multiple signaling pathways could more effectively normalize vessel structure in diseases like diabetic retinopathy.

• Tissue-specific intervention: The variable expression and function of Flt1 across different vascular beds suggests that targeted delivery of Flt1 modulators to specific tissues could achieve therapeutic effects while minimizing systemic complications.

• Timing considerations: Research on developmental Flt1 function indicates critical windows when vascular intervention might be most effective, particularly for diseases with aberrant neovascularization components.

• Biomarker development: Understanding the relationship between circulating sFlt1 levels and vessel abnormalities could lead to biomarkers predicting disease progression or treatment response in conditions ranging from preeclampsia to tumor angiogenesis.