VEGF C Rat (152 a.a.)

What is VEGF-C Rat (152 a.a.) and how does it differ from other VEGF family members?

VEGF-C Rat (152 a.a.) is a recombinant protein containing 152 amino acid residues that belongs to the VEGF growth factor family. This specific variant contains the middle VEGF homology domain and is typically fused with a His-tag (6x His) at the C-terminal end. VEGF-C is most closely related to VEGF-D within the VEGF family, but has distinct receptor binding profiles and biological activities .

Unlike VEGF-A which primarily promotes blood vessel growth, VEGF-C is strongly associated with lymphatic vessel development and maintenance. The rat VEGF-C precursor consists of 416 amino acids, with the middle third comprising the VEGF homology domain and long N- and C-terminal extensions . After processing, the 152 amino acid variant represents a specific form with defined biological activity.

As a member of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family, VEGF-C can influence both angiogenesis and lymphangiogenesis, though its potency for vascular endothelial cells is significantly lower than that of VEGF-A .

What receptors does VEGF-C interact with and what are the functional implications?

VEGF-C interacts with two primary receptors:

• VEGFR-3 (FLT-4): The principal receptor for VEGF-C, predominantly expressed on lymphatic endothelial cells. Binding to VEGFR-3 primarily drives lymphangiogenesis .

• VEGFR-2 (KDR): VEGF-C can also bind to VEGFR-2, which is expressed mainly on blood vascular endothelial cells. This interaction enables VEGF-C to stimulate angiogenesis, though with lower potency than VEGF-A .

It's important to note that VEGF-C undergoes proteolytic processing that affects its receptor binding capabilities. Only fully processed forms of VEGF-C can bind and activate VEGFR-2 receptors, while both processed and unprocessed forms can bind to VEGFR-3 . This differential processing creates a regulatory mechanism that influences whether VEGF-C primarily affects lymphatic or blood vessels in various physiological contexts .

The VEGF-C152S variant (a point mutant where the second conserved cysteine residue is replaced by serine) exclusively activates VEGFR-3 but cannot bind to VEGFR-2. This selective receptor activation makes it useful for studies focused specifically on lymphangiogenesis without concurrent angiogenic effects .

What are the essential methodologies for studying VEGF-C-induced lymphangiogenesis?

Several key methodologies have proven effective for investigating VEGF-C-induced lymphangiogenesis:

• Rat Mesentery Culture Model: This ex vivo system allows observation of both lymphangiogenesis and angiogenesis simultaneously. Mesenteric tissue windows are harvested from adult rats and cultured with VEGF-C, allowing visualization of intact microvascular networks .

Protocol overview:

• Harvest mesenteric tissue windows from adult male rats

• Culture in serum-free minimum essential media with or without VEGF-C (10-200 ng/ml)

• Maintain at 37°C for 3-5 days with daily media changes

• Immunolabel with PECAM (for blood vessels) and LYVE-1 (for lymphatic vessels)

• Quantify lymphatic sprout extensions per vascular area

• Marker Identification: Reliable lymphatic identification requires multiple markers:

  • LYVE-1: Primary lymphatic marker

  • Prox1: Lymphatic endothelial transcription factor

  • Podoplanin: Surface glycoprotein on lymphatic endothelium

  • Reduced PECAM labeling compared to blood vessels

• Filopodia Quantification: Lymphatic filopodia extensions are indicators of active lymphangiogenesis and can be quantified following VEGF-C stimulation .

• Statistical Analysis: Use appropriate statistical methods (ANOVA with Tukey comparison for multiple groups; Student's t-test for two groups) to analyze lymphangiogenic responses to different VEGF-C concentrations or treatment durations .

How does VEGF-C proteolytic processing affect its biological function?

VEGF-C undergoes a complex proteolytic maturation process that significantly impacts its biological activity:

• Initial Synthesis: VEGF-C is synthesized as a pre-pro-protein (416 amino acids in rat) .

• Progressive Processing: During biosynthesis, VEGF-C undergoes removal of both NH₂- and COOH-terminal ends, leaving the central VEGF homology domain (VHD) .

• Functional Consequences:

  • This processing progressively increases VEGF-C's affinity for VEGFR-3

  • Only fully processed forms can bind and activate VEGFR-2 receptors

  • Partially processed forms primarily activate VEGFR-3

• Structural Changes: As a result of glycosylation, processed VEGF-C typically migrates as an 18-24 kDa protein on electrophoretic analysis .

• Dimerization: Recombinant rat VEGF-C lacking the N- and C-terminal extensions forms primarily non-covalently linked dimers, which affects its receptor binding and signaling properties .

This progressive processing creates a regulatory mechanism that determines whether VEGF-C will have predominantly lymphangiogenic effects (via VEGFR-3) or combined lymphangiogenic and angiogenic effects (via both VEGFR-3 and VEGFR-2) .

What is the optimal concentration range for VEGF-C in experimental systems?

Based on established research protocols:

The concentration of 100 ng/ml has been validated in multiple studies and represents a standard working concentration for both in vitro and ex vivo experiments . This concentration reliably induces quantifiable lymphatic sprouting and filopodia formation within 3-5 days in culture systems.

When determining optimal concentration for your specific experimental system, consider:

• Tissue type and accessibility

• Duration of experiment

• Desired endpoint measurements

• Whether you're studying lymphangiogenesis alone or in combination with angiogenesis

How does the VEGF-C152S mutation affect receptor binding and biological function?

The VEGF-C152S mutation represents a significant tool for dissecting the differential effects of VEGF-C on lymphatic versus blood vessels:

• Structural Alteration: VEGF-C152S is generated by replacing the second conserved cysteine residue with a serine residue .

• Receptor Selectivity:

  • Binds and activates VEGFR-3/FLT-4 (lymphatic receptor)

  • Unable to bind or activate VEGFR-2/KDR (vascular receptor)

• Functional Consequences:

  • Inactive in vascular permeability assays

  • Does not increase migration of capillary endothelial cells

  • Maintains lymphangiogenic activity via VEGFR-3 binding

  • Lacks angiogenic activity due to inability to activate VEGFR-2

• Research Applications: This mutant provides an excellent tool for:

  • Isolating lymphangiogenic effects from angiogenic effects

  • Testing the specific contribution of VEGFR-3 signaling

  • Developing targeted lymphatic therapies without vascular side effects

Specifically, the recombinant protein ΔNΔC/VEGF-C/Cys152Ser has been used in studies to selectively regulate lymphangiogenesis through specific binding to VEGFR-3, making it valuable for investigating lymphatic-specific pathways and therapeutic applications .

What temporal relationship exists between lymphangiogenesis and angiogenesis in VEGF-C-stimulated tissues?

Research using the rat mesentery culture model has revealed important temporal dynamics between lymphangiogenesis and angiogenesis:

• Differential Timing: Lymphangiogenesis consistently lags behind angiogenesis in VEGF-C-stimulated tissues, mirroring the pattern observed in vivo .

• Quantitative Assessment: When both processes are monitored simultaneously:

  • Blood capillary sprouting occurs more rapidly

  • Lymphatic sprout extensions develop more gradually

  • Both networks remain intact throughout the culture period (3-5 days)

• Cellular Indicators:

  • LYVE-1-positive filopodia extending from blind-ended lymphatics

  • Filopodia formation along lymphatic vessel segments

  • Number of filopodia significantly increases with VEGF-C treatment

This temporal relationship suggests coordinated but distinct regulatory mechanisms for blood and lymphatic vessel growth, even when stimulated by the same growth factor. Understanding this relationship is crucial for designing experiments with appropriate timepoints and for interpreting results in studies involving both vascular systems .

How can VEGF-C expression and activity be accurately measured in rat tissues?

Several validated methods exist for measuring VEGF-C expression and activity in rat tissues:

• ELISA-Based Quantification:

  • Rat VEGFC solid-phase sandwich ELISA assays are commercially available

  • These assays exclusively recognize both natural and recombinant rat VEGF-C

  • Can be used for serum or cell culture medium samples

  • Employs a matched antibody pair with target-specific antibodies

• Immunohistochemical Detection:

  • Use of specific markers to identify lymphatic vessels:

    • LYVE-1: Primary lymphatic marker

    • Prox1: Lymphatic-specific transcription factor

    • Podoplanin: Surface glycoprotein on lymphatic endothelium

  • Quantify vessel density, branching patterns, or filopodia formation

• Functional Assays:

  • Ex vivo rat mesentery culture model allows quantification of:

    • Lymphatic sprout extensions per vascular area

    • Filopodia formation

    • Endothelial cell proliferation (via BrdU or Ki67 staining)

• Validation Considerations:

  • Sensitivity, specificity, precision, and lot-to-lot consistency are important parameters

  • Controls must be included to ensure reliability of measurements

  • Multiple markers should be used to confirm lymphatic identity

What is the relationship between VEGF-C/VEGFR-3 signaling and salt-related physiological responses?

Recent research has uncovered important connections between VEGF-C signaling and salt homeostasis:

• TonEBP/VEGF-C Pathway: The tonicity-responsive enhancer binding protein (TonEBP)/VEGF-C signaling pathway induces lymphangiogenesis in response to high salt intake, providing a buffering mechanism against salt-induced blood pressure elevation .

• Cardiovascular Effects: In spontaneously hypertensive rats (SHR):

  • High salt diet (8.0% NaCl) upregulates TonEBP expression

  • Increases macrophage infiltration

  • Enhances lymphangiogenesis in left ventricles

• Therapeutic Potential: Experimental manipulation of VEGF-C levels affects cardiovascular outcomes:

  • Overexpression of VEGF-C (using ΔNΔC/VEGF-C/Cys152Ser) leads to:

    • Enhanced cardiac lymphangiogenesis

    • Decreased myocardial fibrosis and macrophage infiltration

    • Preserved left ventricular function

    • Decreased blood pressure after high salt challenge

  • Blocking VEGF-C (using VEGFR-3-Rg) results in:

    • Elevated blood pressure

    • Exacerbated hypertensive left ventricular remodeling

    • Increased fibrosis and macrophage infiltration

    • Diminished lymphangiogenesis

• Mechanistic Insights: VEGF-C appears to attenuate high salt-induced cardiac remodeling through both blood pressure-dependent and -independent mechanisms, highlighting its potential as a therapeutic target in hypertension management .

What experimental models are best suited for studying combined lymphangiogenic and angiogenic effects of VEGF-C?

Several experimental models have been validated for studying the dual effects of VEGF-C:

• Rat Mesentery Culture Model:

  • Advantages:

    • Allows simultaneous observation of lymphangiogenesis and angiogenesis in the same tissue

    • Maintains intact microvascular networks

    • Enables visualization down to single-cell level due to tissue thinness (20-40 μm)

    • Self-contained system that doesn't require embedding into a matrix

    • Simple procedure with clear visualization of network hierarchy

  • Applications:

    • Quantification of both lymphatic and blood vessel sprouting

    • Analysis of temporal relationships between the two processes

    • Testing effects of different VEGF-C concentrations

    • Investigating cellular dynamics in lymphatic/blood vessel patterning

• In Vivo Models:

  • Hypertensive Rat Model:

    • Spontaneously hypertensive rats (SHR) fed high salt diet

    • Allows study of VEGF-C effects on cardiac remodeling

    • Can use retroviral vectors for VEGF-C overexpression or blocking

  • Advantages of in vivo approaches:

    • Preserves physiological context and complex signaling interactions

    • Allows study of long-term effects and systemic responses

    • Enables assessment of functional outcomes (blood pressure, cardiac function)

• Selection Considerations:

  • Research question (mechanism vs. functional outcome)

  • Needed level of cellular resolution

  • Importance of temporal dynamics

  • Requirement for specific manipulation of VEGF-C activity

How can researchers distinguish between direct and indirect effects of VEGF-C in complex tissues?

Distinguishing direct from indirect effects requires sophisticated experimental approaches:

• Receptor-Specific Mutants:

  • VEGF-C152S (Cys152Ser mutation) exclusively activates VEGFR-3 but not VEGFR-2

  • Use this mutant to isolate lymphatic-specific effects from potential blood vessel effects

  • Compare with wild-type VEGF-C to identify differential responses

• Receptor Blocking Strategies:

  • VEGFR-3-Rg fusion protein specifically binds VEGF-C and inhibits lymphangiogenesis

  • Use in combination with receptor-specific antibodies to parse signaling pathways

• Cell-Type Specific Analyses:

  • Use multi-marker immunohistochemistry to identify responding cell populations:

    • LYVE-1, Prox1, podoplanin for lymphatic endothelial cells

    • PECAM for blood vessel endothelium

    • Additional markers for macrophages, fibroblasts, etc.

  • Assess proliferation markers (BrdU, Ki67) in specific cell populations

• Temporal Dissection:

  • Take advantage of the temporal lag between lymphangiogenesis and angiogenesis

  • Analyze early timepoints to capture initial direct effects

  • Monitor progression over time to identify secondary consequences

• Mechanistic Validation:

  • Confirm direct receptor activation through phosphorylation assays

  • Use genetic knockdown approaches in specific cell populations

  • Employ tissue-specific conditional expression systems

What are the current therapeutic applications of VEGF-C/VEGFR-3 manipulation in cardiovascular disease models?

Research has revealed promising therapeutic applications for VEGF-C/VEGFR-3 targeting:

• Hypertension Management:

  • VEGF-C overexpression attenuates high salt intake-induced elevation of blood pressure

  • Enhances lymphatic function, providing a buffering mechanism for excess sodium

• Cardiac Remodeling Prevention:

  • In spontaneously hypertensive rats (SHR), VEGF-C overexpression using retroviral vectors (ΔNΔC/VEGF-C/Cys152Ser) leads to:

    • Enhanced cardiac lymphangiogenesis

    • Decreased myocardial fibrosis

    • Reduced macrophage infiltration

    • Preserved left ventricular function

• Mechanism of Protection:

  • Both blood pressure-dependent effects:

    • Improved sodium/fluid homeostasis

    • Reduced mechanical stress on cardiac tissue

  • Blood pressure-independent effects:

    • Enhanced immune cell clearance

    • Improved tissue drainage

    • Reduced inflammatory signaling

• Potential Therapeutic Strategies:

  • Targeted delivery of VEGF-C to cardiac tissue

  • Development of VEGFR-3-specific agonists with reduced vascular side effects

  • Combination with conventional antihypertensive therapies

  • Prevention of adverse cardiac remodeling in salt-sensitive hypertension

• Challenges and Considerations:

  • Optimal timing of intervention (preventive vs. therapeutic)

  • Tissue-specific targeting to minimize systemic effects

  • Dosing regimens to maintain therapeutic lymphangiogenesis

  • Combination with standard-of-care treatments

How do VEGF-C rat and human variants compare in structure, function, and experimental applications?

Understanding the similarities and differences between rat and human VEGF-C is crucial for translational research:

• Structural Comparison:

  • Rat VEGF-C precursor: 416 amino acids

  • Human VEGF-C precursor: Similar length with high sequence homology

  • The VEGF homology domain is highly conserved between species

  • The rat VEGF-C 152S mutation is analogous to the human VEGF-C 156S mutation

• Functional Homology:

  • Both rat and human VEGF-C bind to VEGFR-3 and VEGFR-2 receptors

  • Both undergo similar proteolytic processing that affects receptor affinity

  • Both primarily stimulate lymphangiogenesis with secondary effects on angiogenesis

  • Species-specific differences in potency may exist but are generally minimal

• Experimental Considerations:

  • For rat models, rat VEGF-C is preferred to avoid potential immunogenicity

  • Human VEGF-C can be used in humanized models or for direct comparison studies

  • Species-specific ELISA assays should be used for accurate quantification

  • Cross-reactivity between species should be verified when using antibodies or inhibitors

• Translational Implications:

  • Findings in rat models using VEGF-C (152 a.a.) can generally inform human applications

  • The conserved VEGF-C152S mutation (rat) and VEGF-C156S mutation (human) provide comparable tools for selective VEGFR-3 activation

  • Careful validation is needed when extending findings across species

What methodological challenges exist in studying VEGF-C-induced tissue remodeling over extended timeframes?

Long-term studies of VEGF-C effects present several methodological challenges:

• Delivery System Limitations:

  • Retroviral vectors can effectively deliver VEGF-C genes but may have variable expression over time

  • Protein stability concerns with direct VEGF-C administration

  • Need for sustained or controlled release systems for consistent effects

• Temporal Monitoring Challenges:

  • Difficulty in non-invasive assessment of lymphatic vessel development

  • Need for serial sampling in longitudinal studies

  • Potential development of compensatory mechanisms over time

• Distinguishing Primary from Secondary Effects:

  • Initial lymphangiogenic effects may trigger cascades of tissue remodeling

  • Changes in blood pressure may cause adaptations independent of direct VEGF-C signaling

  • Inflammatory responses may confound interpretation

• Recommended Approaches:

  • Use inducible expression systems for temporal control

  • Implement multimodal imaging strategies for non-invasive monitoring

  • Establish clear timepoint series with appropriate controls

  • Consider combination of in vivo and ex vivo approaches

  • Use receptor-specific mutants to isolate pathway effects

• Outcome Assessment Standardization:

  • Standardize quantification methods for lymphatic vessel density and functionality

  • Correlate structural changes with functional improvements

  • Integrate molecular, cellular, and physiological endpoints for comprehensive evaluation

What role does VEGF-C play in the crosstalk between inflammation and lymphangiogenesis?

Emerging research highlights important connections between VEGF-C, inflammation, and lymphatic remodeling:

• Macrophage-Lymphatic Interactions:

  • High salt intake increases macrophage infiltration in cardiac tissue of hypertensive rats

  • This infiltration coincides with enhanced lymphangiogenesis and upregulation of TonEBP

  • VEGF-C overexpression decreases macrophage infiltration, suggesting a regulatory feedback loop

• TonEBP/VEGF-C Signaling Pathway:

  • Tonicity-responsive enhancer binding protein (TonEBP) regulates VEGF-C expression

  • Blocking this pathway through mononuclear phagocyte depletion can induce salt-sensitive hypertension

  • This pathway represents a critical link between osmotic stress, inflammation, and lymphatic adaptation

• Therapeutic Implications:

  • VEGF-C-induced lymphangiogenesis may help resolve inflammation by:

    • Enhancing clearance of immune cells and inflammatory mediators

    • Improving tissue drainage to reduce edema

    • Providing routes for antigen presentation and immune regulation

• Experimental Evidence:

  • In spontaneously hypertensive rats, VEGF-C overexpression led to:

    • Decreased myocardial fibrosis

    • Reduced macrophage infiltration

    • Enhanced lymphangiogenesis

    • Preserved cardiac function

  • Conversely, blocking VEGF-C exacerbated inflammation and fibrosis

• Research Directions:

  • Further investigation of the temporal relationship between inflammatory cell recruitment and lymphangiogenesis

  • Exploration of macrophage phenotypes in relation to lymphatic remodeling

  • Development of targeted anti-inflammatory strategies that preserve beneficial lymphangiogenesis