Recombinant Human VEGF-C

Recombinant Human VEGF-C is widely used in research because it is a key regulator of both lymphangiogenesis (formation of lymphatic vessels) and angiogenesis (formation of blood vessels), making it essential for studies involving vascular biology, tissue regeneration, cancer metastasis, and immune modulation.

Key scientific applications and rationale include:

• Lymphangiogenesis Studies: VEGF-C is the principal growth factor for lymphatic endothelial cells (LECs), driving the development and maintenance of the lymphatic system. It is crucial for investigating lymphatic vessel formation, function, and related pathologies such as lymphedema or tumor lymphatic metastasis.

• Angiogenesis Research: While VEGF-C is more specific for lymphatic vessels, it also stimulates blood vessel formation, especially under certain conditions or at higher concentrations. This makes it valuable for studying vascular remodeling, wound healing, and tissue engineering.

• Endothelial Cell Function: Recombinant VEGF-C promotes proliferation, migration, and survival of endothelial cells, both lymphatic and, to a lesser extent, blood vascular. It is commonly used in in vitro assays (e.g., tube formation, migration, proliferation) to dissect signaling pathways and cellular responses.

• Vascular Permeability and Immune Modulation: VEGF-C can increase blood vessel permeability and modulate immune cell trafficking by affecting lymphatic drainage and interstitial fluid balance. This is relevant for studies on inflammation, edema, and neurological injury.

• Therapeutic Modeling: Recombinant VEGF-C is used in preclinical models to test therapies for ischemic injury, tissue regeneration, and lymphatic disorders, as it can induce functional lymphatic and blood vessel networks in vivo.

• Defined, Reproducible Activity: Using recombinant human VEGF-C ensures batch-to-batch consistency, defined molecular structure, and species specificity, which are critical for reproducible experimental results and mechanistic studies.

In summary, recombinant human VEGF-C is a versatile tool for dissecting the molecular and cellular mechanisms of vascular and lymphatic biology, modeling disease, and developing regenerative or anti-metastatic therapies.

Recombinant Human VEGF-C can be used as a standard for quantification or calibration in ELISA assays, provided it is compatible with the antibodies and detection system of your specific assay. This is a common practice in quantitative ELISA protocols for VEGF-C, where recombinant human VEGF-C is used to generate a standard curve against which unknown sample concentrations are measured.

Key considerations:

• Source and Form: The recombinant VEGF-C used as a standard should match the form (e.g., full-length, processed, tagged) recognized by the capture and detection antibodies in your ELISA. Most commercial VEGF-C ELISA kits use recombinant human VEGF-C as the standard, often supplied lyophilized and reconstituted at known concentrations.
• Standard Curve: Prepare a dilution series of the recombinant VEGF-C standard according to your kit or assay protocol to generate a standard curve. This allows interpolation of sample concentrations from the curve.
• Validation: Ensure the recombinant VEGF-C you use is validated for ELISA and is of high purity. Some vendors specifically test their recombinant VEGF-C for use as an ELISA standard.
• Matrix Effects: If you are using a custom ELISA or a kit not supplied with its own standard, confirm that your recombinant VEGF-C behaves similarly to endogenous VEGF-C in your sample matrix (e.g., serum, plasma, cell culture supernatant). Recovery and parallelism studies are recommended to validate this.

Protocol Example:

1. Reconstitute recombinant human VEGF-C to the recommended concentration.
2. Prepare serial dilutions (e.g., 2000 pg/mL to 31.2 pg/mL) in the same buffer as your samples.
3. Add standards and samples to the ELISA plate, proceed with the assay, and measure absorbance.
4. Plot the standard curve and interpolate sample concentrations.

Limitations:

• If your recombinant VEGF-C differs in post-translational modifications or processing from the endogenous form, this may affect antibody recognition and quantification accuracy.
• Always use a new standard curve for each assay run to ensure accuracy.

Summary Table:

In conclusion, recombinant human VEGF-C is widely used as a standard in ELISA assays for quantification, but ensure compatibility with your assay system and validate performance in your specific application.

Recombinant Human VEGF-C has been validated for a range of scientific applications in published research, primarily focused on its roles in lymphangiogenesis, angiogenesis, immune modulation, and therapeutic intervention in disease models.

Key validated applications include:

• In Vivo Therapeutic Studies:

  • Induction of therapeutic lymphangiogenesis in models of acquired lymphedema and chronic postsurgical lymphatic insufficiency.
  • Promotion of angiogenesis in ischemic tissues, such as rabbit hindlimb and corneal models.
  • Amelioration of mesenteric lymphatic drainage, reduction of ascites, and portal pressure in experimental cirrhosis models using nanoformulated VEGF-C.
  • Enhancement of lymphatic drainage of brain-derived fluids and improvement of neurological outcomes after ischemic stroke via intrathecal administration.

• Bioassays and Cell-Based Assays:

  • Stimulation of endothelial cell proliferation and survival, often measured by cell proliferation assays (e.g., HMVEC human microvascular endothelial cells).
  • Activation of VEGFR-3 signaling in lymphatic endothelial cells, assessed by luciferase reporter assays and other cell-based functional assays.
  • Induction of COX-2 expression and angiogenic signaling in leukemic cells via the VEGF-R3/JNK/AP-1 pathway.

• Immunomodulation and Inflammation Models:

  • Expansion of the lymphatic network and reduction of chronic skin inflammation, edema, and inflammatory cell infiltration in mouse models.
  • Modulation of immune cell trafficking, such as increased CD8^+^ T cells and decreased regulatory T cells in mesenteric lymph nodes during cirrhosis.

• Tissue Engineering and Sustained Release:

  • Use in alginate hydrogels for bioactive and sustained release, supporting lymphangiogenic therapeutic applications.

• Gene Expression and Molecular Pathway Studies:

  • Transcriptional profiling of VEGF-C target genes in lymphatic endothelium to identify novel mediators of lymphangiogenesis.
  • Investigation of growth factor signaling in mechanical nociception and endothelial cell lineage specification.

• Disease Models:

  • Application in models of chronic inflammation (psoriasis, arthritis, inflammatory bowel disease), where VEGF-C delivery alleviates disease severity.
  • Studies on vascular permeability and blood vessel formation.

Experimental formats validated:

• In vivo animal models (mouse, rabbit, rat, chicken embryo)
• Whole cell assays (endothelial, leukemic, microvascular cells)
• Bioassays (proliferation, luciferase reporter, binding assays)
• Immunohistochemistry and molecular profiling

Summary Table: Validated Applications of Recombinant Human VEGF-C

These applications demonstrate the versatility of recombinant human VEGF-C in both basic and translational research, particularly in vascular biology, immunology, and regenerative medicine.

To reconstitute and prepare Recombinant Human VEGF-C protein for cell culture experiments, first confirm the specific formulation and recommended protocol from the product’s Certificate of Analysis (CoA) or datasheet, as reconstitution conditions can vary depending on the carrier and buffer used during lyophilization. Below are general best practices based on multiple authoritative sources:

General Reconstitution Guidelines:

• Centrifuge the vial briefly (e.g., 6000–10,000 rpm for 30 seconds) before opening to ensure all lyophilized material is at the bottom.
• Use the recommended solvent:
  • If the protein is lyophilized from PBS (with or without BSA), reconstitute in sterile PBS (pH 7.2–7.4), often at a concentration of 100–250 μg/mL.
  • If the protein is lyophilized from HCl, reconstitute in 4 mM HCl to the recommended concentration (e.g., 250 μg/mL).
  • Some preparations recommend sterile deionized water for reconstitution.
• Add carrier protein if not already present: If the product is carrier-free, add 0.1% human or bovine serum albumin (HSA/BSA) to minimize adsorption and stabilize the protein.
• Mix gently: Swirl or tap the vial gently to dissolve. Avoid vigorous vortexing to prevent protein denaturation.
• Allow the protein to fully dissolve at room temperature for 10–15 minutes, then mix gently again.

Example Protocols:

Storage After Reconstitution:

• Short-term: Store at 2–8°C for up to 1 week.
• Long-term: Aliquot and store at –20°C to –80°C. Avoid repeated freeze-thaw cycles.

Preparation for Cell Culture:

• Dilute the reconstituted stock to the desired working concentration using cell culture medium or sterile PBS with 0.1% HSA/BSA immediately before use.
• Filter sterilize if necessary, using a 0.2 μm filter, especially if the reconstitution buffer or handling is not sterile.
• Typical working concentrations for cell culture range from 1–100 ng/mL, depending on the cell type and experimental design.

Critical Notes:

• Always consult the specific product datasheet or CoA for precise instructions, as buffer composition and recommended concentrations may differ between preparations.
• If the protein is supplied with a carrier (e.g., BSA), additional carrier is usually not needed.
• If the protein is carrier-free, adding a carrier protein is strongly recommended to prevent loss due to adsorption to plasticware.

Summary of Key Steps:

1. Briefly centrifuge the vial.
2. Add the recommended volume of the appropriate buffer (PBS, 4 mM HCl, or sterile water) to achieve the specified concentration.
3. Add 0.1% HSA/BSA if the product is carrier-free.
4. Mix gently until fully dissolved.
5. Aliquot and store appropriately.
6. Dilute to working concentration in cell culture medium just before use.

These steps will help ensure the biological activity and stability of recombinant human VEGF-C for your cell culture experiments.