VEGF C Human

What is the molecular structure of human VEGF-C and how does it differ from other VEGF family members?

Human VEGF-C belongs to the VEGF family but possesses distinct structural characteristics that differentiate it from other members. VEGF-C contains a central VEGF homology domain (VHD) flanked by N- and C-terminal propeptides that undergo proteolytic processing . Unlike VEGF-A, the VHD of VEGF-C contains an extra cysteine residue (Cys137) that affects intermolecular disulfide bond formation and protein stability .

VEGF-C differs from other VEGF family members in its receptor binding profile. While the hemangiogenic VEGFs (VEGF-A, PlGF, and VEGF-B) interact with VEGFR-1, the lymphangiogenic VEGFs (VEGF-C and VEGF-D) interact with VEGFR-3 . VEGF-C can also bind to VEGFR-2, which is expressed on both blood and lymphatic endothelial cells, giving it effects on both vascular systems .

The stepwise proteolytic processing of VEGF-C is a unique feature that distinguishes it from VEGF-A. This processing changes its properties and affinities for receptors throughout its biosynthesis .

How is VEGF-C processed and activated in human tissues?

VEGF-C is initially synthesized as an inactive precursor (pro-VEGF-C) that requires proteolytic processing to become fully active. The activation process involves:

• Secretion of pro-VEGF-C

• Extracellular proteolytic cleavage by ADAMTS3 (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 3)

• Enhancement of this activation by CCBE1 (Collagen And Calcium Binding EGF Domains 1), which acts through two mechanisms:

  • Increasing the processivity of ADAMTS3 enzyme

  • Colocalizing VEGF-C and ADAMTS3 on cell surfaces and extracellular matrix to form a trimeric activation complex

This complex activation mechanism represents a crucial regulatory step for VEGF-C signaling in vivo. The genetic ablation of either CCBE1 or ADAMTS3 in mice results in a general halt of lymphatic development, similar to what is observed with VEGF-C knockout, demonstrating the essential nature of this activation pathway .

Which receptors does human VEGF-C interact with and what signaling pathways does it activate?

Human VEGF-C interacts primarily with two tyrosine kinase receptors:

VEGF-C binding to VEGFR-3 activates several downstream signaling pathways that promote lymphatic endothelial cell proliferation and migration. In humans, VEGFR-3 exists in two splice isoforms: a long form (VEGFR-3l) and a short form (VEGFR-3s) that is exclusive to higher primates . These isoforms activate partially distinct signaling pathways since VEGFR-3s lacks some phosphorylation sites required for mediator docking .

Additionally, engineered variants like VEGF-C(Cys156Ser) have been developed that specifically bind to VEGFR3 homodimers, allowing for more targeted research applications and potential therapeutic interventions .

How do different proteolytic forms of VEGF-C affect its biodistribution and receptor activation potential?

The proteolytic processing of VEGF-C significantly impacts its biological activities and receptor interactions. Research indicates that:

• Pro-VEGF-C (unprocessed form): Exhibits minimal activity and primarily binds to extracellular matrix components, serving as a reservoir that can be activated when needed.

• Partially processed VEGF-C: Shows increased affinity for VEGFR-3 but limited activity on VEGFR-2.

• Fully processed VEGF-C: Demonstrates high affinity for both VEGFR-3 and VEGFR-2, activating both lymphangiogenic and hemangiogenic pathways .

For research applications, it's crucial to consider which form of VEGF-C is being utilized. Most applications to date have not discriminated between different forms of VEGF-C, which may lead to unpredictable results when attempting to engineer lymphatic vessels . The biodistribution of different VEGF-C forms varies significantly due to differences in matrix binding properties and receptor affinities, with consequences for both experimental design and potential therapeutic applications.

What are the optimal experimental conditions for studying VEGF-C-induced lymphangiogenesis in different model systems?

Studying VEGF-C-induced lymphangiogenesis requires careful consideration of experimental conditions across different model systems:

For in vivo studies, the short half-life and systemic side effects of VEGF-C present significant challenges. Recent research has addressed this by developing engineered VEGF-C formulations, such as nanoengineered human recombinant VEGF-C (Cys156Ser) protein encapsulated within nanoscale reverse micelle-based lipocarriers . This approach enhanced gut bioavailability and facilitated targeted delivery to lymphatic vessels when administered orally in rat models of liver cirrhosis .

When designing lymphangiogenesis experiments, researchers should consider:

• The specific form of VEGF-C being utilized

• The presence of necessary cofactors (ADAMTS3, CCBE1)

• The target receptor(s) of interest (VEGFR-3 vs. VEGFR-2)

• The readout systems for measuring lymphangiogenic responses (proliferation, migration, vessel formation)

How does VEGF-C signaling differ between developmental lymphangiogenesis and pathological conditions?

VEGF-C signaling exhibits important differences between developmental lymphangiogenesis and various pathological conditions:

Developmental Context:
During embryogenesis, VEGF-C is constitutively expressed in specific spatiotemporal patterns, with its activation tightly regulated by ADAMTS3 and CCBE1. The genetic knockout of VEGF-C, ADAMTS3, or CCBE1 results in a complete halt of lymphatic development, demonstrating the non-redundant nature of this pathway during development .

Pathological Contexts:
In adult organisms, VEGF-C expression is upregulated in response to various pathological conditions:

• Inflammation: Macrophages potently upregulate VEGF-C expression during inflammation, creating a negative feedback loop in some models while exacerbating the situation in others . This inflammatory-induced VEGF-C may utilize different activation pathways than developmental VEGF-C.

• Liver Cirrhosis: In cirrhotic conditions, mesenteric lymphatic vessels become dilated and dysfunctional. Research has shown that treatment with engineered VEGF-C(Cys156Ser) can restore mesenteric lymphatic vessel function, increase lymphatic vessel proliferation, reduce vessel diameter, and attenuate ascites and portal hypertension in rat models .

• Cancer: VEGF-C signaling in tumors often promotes lymphatic metastasis through mechanisms that may differ from developmental lymphangiogenesis, involving potential evasion of regulatory checkpoints.

These context-dependent differences highlight the importance of understanding the specific regulation of VEGF-C signaling in each experimental or clinical scenario .

What are the most effective methods for recombinant production and purification of human VEGF-C?

Producing recombinant human VEGF-C for research presents several challenges that researchers must address:

Production Approaches:

• Full-length VEGF-C production:

  • Allows for natural proteolytic processing

  • Requires co-expression of processing enzymes (ADAMTS3) and cofactors (CCBE1)

  • Results in heterogeneous products requiring complex purification strategies

• Truncated VEGF-C production (mature domain only):

  • Bypasses proteolytic processing requirements

  • Faces challenges due to the extra cysteine residue (Cys137) in the VEGF homology domain

  • Requires optimization to ensure proper disulfide bond formation and protein stability

• VEGF-C variant production (e.g., VEGF-C Cys156Ser):

  • Offers receptor specificity (preferential binding to VEGFR-3)

  • May provide better stability and homogeneity

  • Used in targeted therapeutic applications

Purification Considerations:

• Affinity chromatography using receptor-based columns

• Size exclusion chromatography to separate different proteolytic forms

• Activity assays to confirm biological function

• Endotoxin removal for in vivo applications

When working with the Cys156Ser variant, researchers have successfully developed stable formulations by encapsulating the protein within nanoscale reverse micelle-based lipocarriers, which enhances stability and facilitates targeted delivery to lymphatic vessels .

How can researchers accurately measure VEGF-C-induced lymphangiogenesis in experimental models?

Accurately measuring VEGF-C-induced lymphangiogenesis requires multi-parameter assessment approaches:

In Vitro Assessment Methods:

• Proliferation assays: Quantifying LEC proliferation using BrdU incorporation, Ki67 staining, or cell counting

• Migration assays: Scratch wound healing or transwell migration assays

• Tube formation assays: 2D Matrigel assays or 3D spheroid sprouting assays

• Receptor phosphorylation: Measuring VEGFR-3 phosphorylation by immunoprecipitation and western blotting

In Vivo Assessment Methods:

• Lymphatic vessel visualization and quantification:

  • Immunohistochemical staining for lymphatic markers (Pdpn, LYVE-1, VEGFR-3)

  • Quantifying vessel number, diameter, and density

• Functional assessment of lymphatic drainage:

  • Tracer dye injection and tracking

  • Measurement of interstitial pressure

  • Assessment of fluid or macromolecule clearance from tissues

• Molecular analysis of lymphatic endothelial cells:

  • Gene expression analysis of isolated LECs

  • Quantification of inflammatory markers (e.g., eNOS, iNOS)

  • Single-cell sequencing to identify LEC subpopulations

What strategies can optimize VEGF-C delivery for therapeutic lymphangiogenesis applications?

Optimizing VEGF-C delivery for therapeutic lymphangiogenesis requires addressing several challenges, including short half-life, systemic side effects, and targeted delivery to specific tissues:

Advanced Delivery Strategies:

• Nanoformulation approaches:

  • Engineered VEGF-C encapsulated in nanoscale reverse micelle-based lipocarriers

  • Enhances stability and gut bioavailability

  • Facilitates targeted delivery to lymphatic vessels

  • Demonstrated efficacy in rat models of liver cirrhosis when delivered orally

• Controlled release systems:

  • Hydrogels incorporating VEGF-C for sustained release

  • Biodegradable microspheres or scaffolds

  • Co-delivery with stabilizing factors or cofactors (e.g., CCBE1)

• Targeted delivery approaches:

  • Tissue-specific targeting ligands

  • Exploitation of natural lymphatic uptake pathways

  • Local administration at sites requiring lymphatic regeneration

Dosing Considerations:
In experimental models, E-VEGF-C has been administered orally at a dose of 600 μg/kg body weight on alternate days for 2 weeks, showing significant effects on mesenteric lymphatic vessel proliferation and function without adverse effects . This dosing regimen resulted in a biphasic serum peak, with the first peak appearing at approximately 10 minutes and the second peak at approximately 5 hours after administration .

Monitoring Strategies:

• Pharmacokinetic analysis to track biodistribution

• Lymphatic-specific imaging techniques

• Functional assessment of lymphatic drainage

• Biomarkers of lymphangiogenic response

These optimization strategies are essential for translating VEGF-C-based therapies from preclinical models to clinical applications for conditions involving lymphatic dysfunction .

What are the current knowledge gaps in understanding VEGF-C regulation and signaling?

Despite significant advances in VEGF-C research, several important knowledge gaps remain:

• Regulation of VEGF-C expression: Compared to VEGF-A, relatively little is known about the regulation of VEGF-C expression in vivo. While inflammation is known to upregulate VEGF-C expression, particularly in macrophages, the detailed molecular mechanisms and signaling pathways involved remain incompletely understood .

• Regulation of VEGF-C activation factors: While the activation mechanism of VEGF-C by ADAMTS3 and CCBE1 has been extensively studied, virtually nothing is known about the regulation of ADAMTS3 expression and activity or the regulation of CCBE1 in different physiological and pathological contexts .

• Context-dependent signaling outcomes: The specific signaling pathways and biological outcomes triggered by VEGF-C in different cellular contexts and disease states require further investigation. For example, how VEGF-C signaling differs between developmental lymphangiogenesis, inflammatory conditions, and various pathological states remains to be fully elucidated.

• Receptor trafficking and recycling: The dynamics of VEGFR-3 trafficking, recycling, and degradation following VEGF-C binding, and how these processes influence signaling duration and intensity, represent important areas for future research.

• Cross-talk with other signaling pathways: The interactions between VEGF-C/VEGFR-3 signaling and other important signaling pathways in lymphatic endothelial cells need further exploration to understand the integrated cellular responses to multiple stimuli.

How can engineered variants of VEGF-C advance lymphatic tissue engineering applications?

Engineered variants of VEGF-C hold significant promise for advancing lymphatic tissue engineering:

Recent research demonstrates the potential of engineered VEGF-C variants, such as the successful use of nanoformulated VEGF-C(Cys156Ser) to restore mesenteric lymphatic vessel function in rat models of liver cirrhosis . This approach significantly increased lymphatic vessel proliferation, improved lymph drainage, and attenuated ascites and portal hypertension .

What potential therapeutic applications of VEGF-C are emerging beyond established lymphedema treatments?

Research indicates several promising emerging therapeutic applications for VEGF-C beyond established lymphedema treatments:

• Liver cirrhosis and portal hypertension: Recent studies demonstrate that engineered VEGF-C (E-VEGF-C) administration significantly increased mesenteric lymphatic vessel proliferation, improved lymph drainage, and attenuated abdominal ascites and portal pressures in rat models of decompensated liver cirrhosis . This suggests potential applications in managing complications of cirrhosis.

• Inflammatory bowel diseases: VEGF-C therapy has shown efficacy in improving lymphatic function and ameliorating inflammation in inflammatory bowel disease models, representing a potential new treatment approach for these conditions .

• Rheumatoid arthritis: Enhancing lymphangiogenesis with VEGF-C has demonstrated anti-inflammatory effects in models of rheumatoid arthritis, potentially offering a novel therapeutic strategy .

• Skin inflammation and wound healing: VEGF-C-induced lymphangiogenesis may accelerate wound healing and reduce inflammation in skin conditions, suggesting applications in dermatology and wound care.

• Hepatic encephalopathy: VEGF-C therapies aimed at enhancing lymphangiogenesis have shown efficacy in improving lymphatic function in models of hepatic encephalopathy .

• Lymphatic targeting for drug delivery: The lymphatic system's role in immune cell trafficking and antigen presentation makes it an attractive target for immunomodulatory therapies. VEGF-C-induced lymphangiogenesis could enhance lymphatic drug delivery for vaccines or immunotherapies.

These emerging applications highlight the growing recognition of lymphatic vessels as important therapeutic targets across a wide range of diseases, with VEGF-C playing a central role in modulating lymphatic function and associated pathologies .