VEGF C Rat

What is the molecular structure and function of rat VEGF-C?

Rat VEGF-C is a member of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family that plays crucial roles in both angiogenesis and lymphangiogenesis. The protein undergoes complex proteolytic maturation to generate multiple processed forms. These processed forms primarily bind and activate VEGFR-3 receptors, while only the fully processed form can bind and activate VEGFR-2 receptors . Rat VEGF-C is structurally and functionally similar to vascular endothelial growth factor D, with a gene ID of 114111 in rats .

The protein's primary functions include promoting endothelial cell growth, influencing blood vessel permeability, and stimulating lymphatic vessel formation. Unlike other VEGF family members that predominantly affect blood vessels, VEGF-C demonstrates dual functionality by affecting both blood and lymphatic vasculature, with particularly strong effects on lymphatic endothelial cells through the VEGFR-3 signaling pathway.

How does rat VEGF-C differ from human VEGF-C in experimental settings?

While rat and human VEGF-C share significant homology and functional similarities, researchers should note several important experimental considerations. The species-specific binding affinities to receptors can impact cross-species studies. When using recombinant human VEGF-C (Cys156Ser) in rat models, as demonstrated in several studies, the protein remains bioactive and produces measurable lymphangiogenic effects .

The protein's proteolytic processing may have subtle species-specific differences affecting its biological activity. In experimental settings, human recombinant VEGF-C (particularly the Cys156Ser variant) has been successfully used in rat models at concentrations of 600 μg/kg when delivered orally in specialized formulations . Traditional experimental designs have used concentrations of 100 ng/ml for in vitro and ex vivo studies to effectively stimulate lymphatic endothelial cells and induce lymphangiogenesis .

What are the primary signaling pathways activated by VEGF-C in rat models?

VEGF-C primarily signals through the VEGFR-3 pathway in lymphatic endothelial cells, activating downstream pathways that promote lymphangiogenesis. The VEGF-C/VEGFR-3 axis is particularly prominent in intestinal lymphangiogenesis and inflammation-induced lymphatic vessel growth . In rat models, VEGFR-3 expression is significantly increased during pathological conditions such as cirrhosis, indicating upregulation of this pathway during disease states .

Additionally, VEGF-C can activate VEGFR-2 in its fully processed form, which contributes to its angiogenic effects on blood vessels. The protein also upregulates the expression of vascular endothelial-cadherin in lymphatic endothelial cells, which functionally improves the permeability characteristics of these cells . This dual receptor activation explains why VEGF-C treatment results in both blood vessel sprouting (via VEGFR-2) and lymphatic vessel development (primarily via VEGFR-3).

What are the most reliable methods for quantifying VEGF-C in rat samples?

The sandwich ELISA (enzyme-linked immunosorbent assay) is the gold standard for quantifying rat VEGF-C in biological samples. This method uses a target-specific antibody pre-coated in microplate wells to capture VEGF-C, followed by addition of a detector antibody and substrate solution that produces a measurable signal proportional to VEGF-C concentration .

Validated ELISA kits for rat VEGF-C can accurately quantify the protein in rat serum or cell culture medium, with excellent specificity for both natural and recombinant rat VEGF-C . When selecting detection methods, researchers should consider the following recovery rates for recombinant rat VEGF in different sample types:

These recovery rates indicate high reliability across different sample types, with particularly excellent recovery in citrate plasma samples .

How can researchers effectively visualize VEGF-C-induced lymphangiogenesis in rat tissues?

Visualization of VEGF-C-induced lymphangiogenesis in rat tissues requires specific immunostaining techniques targeting lymphatic markers. The most effective approach combines immunofluorescence staining for multiple lymphatic markers:

• LYVE-1 (Lymphatic Vessel Endothelial Hyaluronan Receptor 1): Primary marker for lymphatic vessels

• Prox1: Transcription factor specific to lymphatic endothelium

• Podoplanin (Pdpn): Transmembrane glycoprotein highly expressed in lymphatic vessels

• VEGFR-3: Receptor for VEGF-C, enriched on lymphatic endothelium

For functional assessment of lymphatic vessels, researchers can use tracer dye experiments. For example, BODIPY FL-C16 can be administered via gavage to assess drainage and leakage of mesenteric lymphatic vessels . Increased fluorescence inside dilated lymphatic vessels indicates impaired drainage, while fluorescence outside vessels indicates leakage. The effectiveness of VEGF-C treatment can be measured by reduced fluorescence both inside (improved drainage) and outside (reduced leakage) the lymphatic vessels .

What controls should be included when measuring VEGF-C expression in rat experimental models?

When measuring VEGF-C expression in rat experimental models, researchers should include several critical controls:

• Positive and negative tissue controls: Include tissues known to express high levels of VEGF-C (positive control) and tissues with minimal expression (negative control)

• Antibody validation controls: For immunohistochemistry or Western blot, include primary antibody omission controls and isotype controls to assess non-specific binding

• Reference genes for qPCR: When measuring VEGF-C mRNA expression, use multiple validated reference genes for normalization (GAPDH, β-actin, and 18S rRNA are commonly used)

• Vehicle controls: When studying VEGF-C treatments (like the E-VEGF-C nanoformulation), include appropriate vehicle controls (e.g., nanoformulation without VEGF-C)

• Recombinant protein standards: For quantitative assays, include a standard curve using recombinant rat VEGF-C at known concentrations

• Treatment timing controls: Given that lymphangiogenesis lags angiogenesis in response to VEGF-C treatment, include multiple time points to capture the complete biological response

How effective is the rat mesentery culture model for studying VEGF-C-induced lymphangiogenesis?

The rat mesentery culture model has proven highly effective for studying VEGF-C-induced lymphangiogenesis, offering several unique advantages:

• It maintains intact lymphatic and blood microvascular networks after culture for 3-5 days, with viable vascular cells along the networks

• It allows simultaneous observation of both angiogenesis and lymphangiogenesis in the same microvascular network, revealing that lymphangiogenesis lags angiogenesis in response to VEGF-C stimulation

• VEGF-C treatment (100 ng/ml) significantly increases lymphatic sprout extensions after five days of culture, and increases the number of filopodia, which are associated with active lymphangiogenesis

• The model enables clear identification of lymphatic vessels based on decreased PECAM labeling, increased vessel diameters, and positive labeling for LYVE-1, Prox1, and Podoplanin

• It allows for quantification of both blood capillary sprouts and lymphatic sprout extensions per vascular area, providing quantitative metrics for assessing lymphangiogenic responses

This ex vivo model bridges the gap between in vitro cell culture and in vivo animal models, offering controlled conditions while maintaining the complexity of intact microvascular networks.

What are the optimal concentrations and delivery methods for VEGF-C in different rat experimental models?

The optimal concentrations and delivery methods for VEGF-C vary by experimental model:

Ex Vivo Mesentery Culture Model:

• Optimal concentration: 100 ng/ml VEGF-C in serum-free media

• Rationale: This concentration has been validated to stimulate lymphatic endothelial cells and induce lymphangiogenesis

• Duration: 3-5 days of culture to observe both angiogenic and lymphangiogenic effects

In Vivo Cirrhotic Rat Models:

• Formulation: Engineered VEGF-C (E-VEGF-C) nanoformulation using recombinant human VEGF-C (Cys156Ser)

• Delivery method: Oral administration

• Dosage: 600 μg/kg body weight

• Administration schedule: Alternate days for up to 2 weeks

• Nanoparticle characteristics: Mean particle size of 134.8 ± 0.47 nm, polydispersity index of 0.126 ± 0.01, zeta potential of −21.9 ± 1.24 mV

The nanoformulation approach significantly enhances VEGF-C delivery to target tissues, with spectrofluorimetry and fluorescence microscopy confirming increased signal in mesenteric tissues 2 hours after oral administration . This delivery method overcomes traditional challenges with protein therapeutics by protecting VEGF-C from degradation and enhancing its uptake by lymphatic endothelial cells.

How does VEGF-C treatment affect lymphatic vessel morphology and function in rat disease models?

VEGF-C treatment produces significant changes in lymphatic vessel morphology and function in rat disease models, particularly in cirrhosis:

Morphological Changes:

• Increased number of podoplanin-positive mesenteric lymphatic vessels

• Significantly reduced vessel diameter (normalizing the pathological dilation seen in cirrhosis)

• Increased sprouting of new lymphatic vessels from existing ones

• Increased branching points of mesenteric lymphatic vessels close to the intestine

Functional Improvements:

• Enhanced lymphatic drainage capacity (measured by reduced BODIPY tracer retention)

• Decreased lymphatic vessel leakage (measured by reduced extravascular tracer)

• Improved vessel integrity and reduced permeability (through upregulation of vascular endothelial-cadherin)

These changes collectively result in physiological improvements, including reduced ascites and portal pressures in cirrhotic rats. The treatment also modifies immune cell composition in mesenteric lymph nodes, with increases in CD8+CD134+ T cells and decreases in CD25+ regulatory T cells, suggesting enhanced immune surveillance .

How does VEGF-C treatment influence immune cell trafficking in rat lymphatic systems?

VEGF-C treatment significantly alters immune cell trafficking and composition in rat lymphatic systems, with important implications for immune function:

In E-VEGF-C treated cirrhotic rats, mesenteric lymph nodes (MLNs) show increased CD8+CD134+ T cells and decreased CD25+ regulatory T cells compared to vehicle-treated controls . This shift suggests enhanced effector T cell activation and reduced immunosuppressive signals.

The improved lymphatic vessel function following VEGF-C treatment enhances antigen trafficking to lymph nodes, potentially improving immune surveillance. This is evidenced by limited bacterial translocation to MLNs in E-VEGF-C treated rats compared to vehicle-treated controls .

The treatment reduces endotoxin levels in ascites and blood, indicating improved containment of bacterial products within the intestinal and lymphatic compartments . This has important implications for reducing systemic inflammation in disease models.

These immunomodulatory effects demonstrate that VEGF-C therapy not only affects vascular morphology but also influences the immune microenvironment, highlighting the interconnection between the lymphatic system and immune function.

What is the temporal relationship between VEGF-C-induced angiogenesis and lymphangiogenesis in rat models?

Research using the rat mesentery culture model has revealed a distinct temporal relationship between VEGF-C-induced angiogenesis and lymphangiogenesis:

• Angiogenesis (blood vessel sprouting) occurs first, with significant increases observed by day 3 of VEGF-C treatment

• Lymphangiogenesis (lymphatic vessel sprouting) follows later, becoming significant by day 5 of VEGF-C treatment

• This temporal lag mirrors the in vivo developmental pattern, where blood vessels typically form before lymphatic vessels during embryogenesis

This sequential response has important experimental design implications, as studies focusing on lymphangiogenesis must allow sufficient time (5+ days) for observable effects, while angiogenic responses can be detected earlier. The mechanisms behind this temporal difference likely relate to different threshold requirements for VEGFR-2 versus VEGFR-3 activation or differences in downstream signaling kinetics between blood and lymphatic endothelial cells.

How can researchers distinguish between direct VEGF-C effects and secondary inflammation-induced lymphangiogenesis?

Distinguishing between direct VEGF-C effects and secondary inflammation-induced lymphangiogenesis requires careful experimental design and multiple analytical approaches:

Experimental Approaches:

• Receptor specificity analysis: Use VEGFR-3 selective inhibitors (like SAR) to ablate lymphatic vessels, which can help determine if observed effects are directly mediated through VEGFR-3 signaling

• Gene expression profiling: Measure inflammatory markers (CCL21, COX2, eNOS) alongside VEGF-C/VEGFR-3 to assess whether inflammation precedes or follows lymphangiogenesis

• Time-course studies: Observe the sequence of inflammatory marker expression versus lymphangiogenic responses to determine causality

• Selective depletion studies: Use anti-inflammatory agents to suppress inflammation and observe if lymphangiogenesis is impaired

Key Indicators of Inflammation-Induced Lymphangiogenesis:

• Upregulation of inflammatory cytokines and chemokines prior to lymphatic vessel proliferation

• Presence of inflammatory cell infiltrates near proliferating lymphatic vessels

• Increased expression of adhesion molecules on lymphatic endothelium

• Correlation between severity of inflammation and degree of lymphangiogenesis

In cirrhotic rat models, researchers have observed increased expression of inflammatory markers (CCL21, COX2, eNOS) alongside VEGF-C upregulation, suggesting a complex interplay between inflammation and lymphangiogenesis .

What therapeutic potential does VEGF-C show in rat models of lymphatic dysfunction?

VEGF-C demonstrates significant therapeutic potential in rat models of lymphatic dysfunction, particularly in cirrhosis and portal hypertension:

In cirrhotic rats, treatment with engineered VEGF-C (E-VEGF-C) produced several beneficial effects:

• Significantly reduced ascites accumulation

• Decreased portal pressure

• Improved mesenteric lymphatic vessel drainage

• Prevention of lymphatic vessel leakage

• Reduced bacterial translocation and endotoxemia

These benefits were observed across multiple cirrhosis models (CCl4, TAA, and BDL), suggesting broad applicability regardless of etiology. Additionally, E-VEGF-C treatment showed efficacy in non-cirrhotic portal hypertension (PPVL model), indicating potential applications beyond cirrhosis .

The mechanism appears to involve VEGF-C-induced proliferation of functional lymphatic vessels that improve drainage of excess fluid and potentially harmful substances like bacteria and their products. The upregulation of vascular endothelial-cadherin in lymphatic endothelial cells also contributes to improved vessel integrity and reduced leakage .

What are the optimal methods for engineering VEGF-C delivery systems in rat research models?

Engineering optimal VEGF-C delivery systems for rat research models requires careful consideration of formulation, characterization, and delivery route:

Nanoformulation Development:

• Use high-pressure homogenization/microfluidization techniques to prepare reverse micelles (RMs)

• Incorporate recombinant human VEGF-C (Cys156Ser) into prepared RMs

• Encapsulate inside lipocarriers prepared with distearoyl-rac-glycerol-PEG2K

Critical Nanoparticle Parameters:

• Mean particle size: 134.8 ± 0.47 nm (optimal for lymphatic uptake)

• Polydispersity index: 0.126 ± 0.01 (indicating uniform particle size distribution)

• Zeta potential: −21.9 ± 1.24 mV (providing colloidal stability)

• pH value: 6.369 ± 0.004 (compatible with biological systems)

Release Profile:

• Initial burst release: 31.95 ± 1.52% VEGF-C at 2 hours

• Peak release: 84.66 ± 1.82% at 4 hours

• Follows zero-order kinetics (concentration-independent release)

Delivery Route:
For targeting mesenteric lymphatic vessels, oral administration has proven effective with biodistribution studies confirming intense fluorescence signal in mesenteric tissues 2 hours after administration . This approach leverages the natural uptake of lipid-based particles by intestinal lymphatics.

The engineered formulation shows excellent cellular uptake by lymphatic endothelial cells both in vitro and in vivo, demonstrating that this delivery system effectively targets the intended lymphatic tissues .

How do the effects of VEGF-C compare with other lymphangiogenic factors in rat experimental models?

While the search results don't provide direct comparisons between VEGF-C and other lymphangiogenic factors in rat models, several key considerations can guide researchers:

VEGF-C has demonstrated specificity for lymphatic vessels through its predominant binding to VEGFR-3, particularly the Cys156Ser variant used in several studies . This receptor specificity distinguishes it from factors that may have broader effects on both blood and lymphatic vessels.

The dual effects of VEGF-C on promoting both lymphatic vessel proliferation and functional improvement (reduced diameter, improved drainage, decreased leakage) represent a comprehensive therapeutic profile that may not be matched by other single factors .

Researchers should consider that while other VEGF family members (particularly VEGF-D) share some lymphangiogenic properties, VEGF-C appears to have the strongest and most consistent lymphangiogenic effects in experimental models .

Future comparative studies should evaluate VEGF-C against other lymphangiogenic factors such as VEGF-D, angiopoietins, and hepatocyte growth factor, assessing not only vessel growth but also functional improvements in drainage, permeability, and immune cell trafficking.

What are common challenges in detecting VEGF-C expression in rat tissues and how can they be overcome?

Researchers face several challenges when detecting VEGF-C expression in rat tissues:

Challenge 1: Low baseline expression levels

• Solution: Use sensitive detection methods like ELISA with appropriate sample concentration or amplification steps

• Implementation: Select validated ELISA kits with documented sensitivity for rat VEGF-C with recovery rates above 90% for most sample types

Challenge 2: Cross-reactivity with other VEGF family members

• Solution: Use antibodies specifically validated for rat VEGF-C with minimal cross-reactivity

• Implementation: Verify antibody specificity by testing against recombinant VEGF-A, VEGF-B, and VEGF-D

Challenge 3: Tissue-specific expression patterns

• Solution: Use proper tissue collection, processing, and storage protocols to preserve VEGF-C integrity

• Implementation: Process tissues rapidly, use appropriate fixatives for immunohistochemistry, and optimize extraction buffers for protein/RNA isolation

Challenge 4: Distinguishing between different processed forms of VEGF-C

• Solution: Use Western blotting with appropriate antibodies that can detect different processed forms

• Implementation: Include positive controls with known molecular weights of different VEGF-C forms

Challenge 5: Variable expression in disease models

• Solution: Include appropriate time course studies and multiple tissue sampling locations

• Implementation: Sample tissues at different disease stages and from multiple anatomical locations to capture the full expression pattern

How can researchers optimize lymphatic vessel visualization in rat VEGF-C studies?

Optimizing lymphatic vessel visualization in rat VEGF-C studies requires specialized techniques:

Immunofluorescence Multi-marker Approach:

• Use combinations of lymphatic markers for definitive identification:

  • LYVE-1: Primary marker for lymphatic vessels

  • Prox1: Nuclear transcription factor specific to lymphatic endothelium

  • Podoplanin: Membrane marker for lymphatic endothelial cells

  • VEGFR-3: Receptor concentrated on lymphatic endothelium

Whole-mount Staining Technique:

• For tissues like mesentery, use whole-mount staining rather than thin sections

• This preserves the three-dimensional architecture of lymphatic networks

• Allows visualization of sprouting, branching, and connections between vessels

Functional Visualization Approaches:

• Use tracer dyes like BODIPY FL-C16 administered via gavage

• Image at appropriate timepoints (e.g., 2 hours post-administration)

• Quantify fluorescence both inside vessels (for drainage assessment) and outside vessels (for leakage assessment)

Digital Analysis Optimization:

• Use standardized imaging parameters across experimental groups

• Employ software analysis to quantify vessel metrics:

  • Vessel number per area

  • Vessel diameter

  • Branching points

  • Sprout extensions

These combined approaches provide both structural and functional insights into VEGF-C effects on the lymphatic vasculature.

What factors influence VEGF-C activity in rat experimental models and how can they be controlled?

Multiple factors influence VEGF-C activity in rat experimental models that researchers should control:

Proteolytic Processing:

• VEGF-C requires proteolytic processing to generate fully active forms

• Control: Use pre-processed recombinant VEGF-C variants (e.g., Cys156Ser) that don't require further processing

Receptor Expression Levels:

• VEGFR-3 expression varies between tissues and disease states

• Control: Characterize baseline VEGFR-3 expression in target tissues before VEGF-C administration

• Note: Cirrhotic rats show significantly increased VEGFR-3 expression compared to controls

Delivery System Characteristics:

• Nanoparticle size, charge, and composition affect VEGF-C delivery and activity

• Control: Standardize particle characteristics (size ~135 nm, polydispersity index ~0.13, zeta potential ~-22 mV)

• Monitor: Check VEGF-C release kinetics from delivery systems (peak release typically at 4 hours)

Disease State:

• Underlying pathologies affect baseline lymphatic function and responsiveness

• Control: Use appropriate disease models (CCl4, TAA, BDL for cirrhosis) with standardized induction protocols

• Include: Disease severity assessments (e.g., serum ALT, albumin levels)

Treatment Timing and Duration:

• Different models show varied response kinetics to VEGF-C

• Control: Design time-course studies to determine optimal treatment duration (typically 2 weeks of alternate-day dosing in cirrhosis models)

• Remember: Lymphangiogenesis lags angiogenesis, requiring longer treatment periods for full effect

By controlling these factors, researchers can achieve more consistent and interpretable results in VEGF-C studies.