Recombinant Human Angiopoietin-4

Using Recombinant Human Angiopoietin-4 (rhAng-4) in research applications is valuable for studying and modulating vascular biology, angiogenesis, endothelial barrier function, and tissue repair, as well as for modeling and potentially treating diseases involving vascular dysfunction and inflammation.

Key scientific reasons to use rhAng-4 include:

• Vascular Permeability and Barrier Protection: rhAng-4 has been shown to protect and restore endothelial and epithelial barrier integrity, reducing permeability and maintaining tight junction proteins in models of ischemia/reperfusion injury and inflammation. This makes it a useful tool for studying mechanisms of barrier dysfunction and for developing therapies targeting vascular leakage or tissue edema.

• Angiogenesis and Vessel Maturation: rhAng-4 promotes angiogenesis (formation of new blood vessels) and supports vessel maturation and stabilization, which is critical in wound healing, tissue regeneration, and cardiovascular research. It can be used to model neovascularization in vitro and in vivo, and to dissect pathways involved in endothelial cell function.

• Inflammation Modulation: ANGPTL4 modulates inflammatory responses, including reducing oxidative stress and apoptosis in tissues subjected to injury. This property is relevant for research into inflammatory diseases, tissue injury, and repair mechanisms.

• Metabolic Regulation: ANGPTL4 is a key regulator of lipid metabolism through inhibition of lipoprotein lipase (LPL), impacting energy homeostasis and metabolic disease models. Recombinant protein can be used to probe these pathways in cell and animal models.

• Disease Modeling and Therapeutic Development: Dysregulation of ANGPTL4 is implicated in kidney diseases, cardiovascular disorders, cancer, and retinopathies. Using rhAng-4 allows for mechanistic studies and preclinical testing of therapeutic strategies targeting these conditions.

• Wound Healing and Tissue Repair: The protein accelerates wound closure, improves collagen architecture, and enhances the biomechanical quality of healed tissues, making it relevant for regenerative medicine and dermatological research.

• Biomarker and Diagnostic Research: ANGPTL4 is being explored as a biomarker for diseases such as diabetic kidney disease and nephrotic syndrome, and recombinant protein is essential for assay development and validation.

In summary, recombinant human Angiopoietin-4 is a versatile reagent for dissecting vascular, metabolic, and inflammatory pathways, modeling disease, and developing new therapeutic approaches in a wide range of biomedical research fields.

Yes, recombinant human Angiopoietin-4 can be used as a standard for quantification and calibration in ELISA assays. This is a well-established practice in the field.

Standard Curve Development

Recombinant human Angiopoietin-4 protein serves as the reference standard for constructing calibration curves in sandwich ELISA formats. The standard is typically reconstituted according to manufacturer specifications and then serially diluted to create a concentration range that brackets your expected sample values.

Typical Concentration Ranges

Standard curves are commonly prepared using recombinant Angiopoietin-4 at concentrations spanning multiple orders of magnitude. Representative dilution series include concentrations such as 0.19, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, and 25 ng/mL, or alternatively 18.7 to 1200 pg/mL depending on the assay sensitivity requirements.

Assay Performance Characteristics

When using recombinant Angiopoietin-4 as your standard, you can expect:

• Detection ranges typically spanning 0.78–50 ng/mL or 3.125–100 ng/mL, depending on the specific assay configuration
• High specificity with minimal cross-reactivity to structural analogs
• Sensitivity with lowest detectable doses below 0.781 ng/mL
• Precision with intra-batch variation coefficients less than 10% and inter-batch variation less than 15%

Practical Considerations

When preparing your standard curve, reconstitute the stock standard with the appropriate diluent provided with your kit and allow adequate equilibration time before use. Ensure all standards fall within your assay's validated detection range, and dilute your samples appropriately if their concentrations exceed the highest standard concentration.

Recombinant human angiopoietin-like protein 4 (rhANGPTL4) has been validated for numerous applications across diverse biological and clinical research contexts.

Barrier Protection and Tissue Repair

Intestinal barrier integrity represents one of the most thoroughly validated applications. rhANGPTL4 has demonstrated significant protective effects in intestinal ischemia-reperfusion injury models, reversing indicators of barrier dysfunction including increased permeability, loss of tight junction proteins (zonula occludens-1, claudin-2), and VE-cadherin disruption. The protein counteracts inflammatory cascades, oxidative stress, excessive autophagy, and apoptosis in intestinal epithelial cells and vascular endothelial cells.

Blood-brain barrier protection has also been established as a validated application. In ischemic stroke models, recombinant human ANGPTL4 decreased VEGFR2–VE-cadherin complex disruption via Src signaling, thereby increasing endothelial cell barrier integrity in cerebral microcirculation.

Angiogenesis and Vascular Function

rhANGPTL4 promotes in vitro tube formation of immortalized human microvascular endothelial cells and stimulates neovascularization in vivo. The protein has been validated for promoting vessel integrity and stabilizing endothelial cells following myocardial infarction and ischemia. When delivered via hydrogel patches providing sustained levels within cardiac tissue, rhANGPTL4 enhanced vascularization and promoted recruitment of M2 macrophages.

Wound Healing

Accelerated wound re-epithelialization in diabetic models has been demonstrated, with recombinant ANGPTL4 stimulating wound angiogenesis through keratinocyte-to-endothelial cell communication. The protein regulates angiogenesis via integrin/JAK/STAT3-mediated upregulation of inducible nitric oxide synthase expression in wound epithelia. Additionally, the C-terminal fragment modulates cell adhesion through interactions with heparan sulfate proteoglycans, integrins, vitronectin, and fibronectin, thereby promoting keratinocyte migration.

Metabolic Regulation

Glucose homeostasis and insulin sensitivity represent validated applications. Adenovirus-mediated expression of ANGPTL4 potently decreased blood glucose and improved glucose tolerance. In diabetic mouse models, ANGPTL4 treatment reduced hyperglycemia to normal levels and markedly alleviated glucose intolerance and hyperinsulinemia.

Analytical Applications

rhANGPTL4 has been validated for use as an ELISA standard and immunoassay standard, with applications in measuring plasma ANGPTL4 levels in both diabetic and non-diabetic subjects. The protein also serves as an SDS-PAGE control in protein analysis workflows.

Biomarker Development

Plasma ANGPTL4 has emerged as a novel biomarker capable of predicting 10-year mortality and enhancing risk prediction beyond established cardiovascular risk factors.

To reconstitute and prepare Recombinant Human Angiopoietin-4 (ANGPT4) protein for cell culture experiments, follow these general best practices based on manufacturer guidelines and scientific recommendations:

Reconstitution

1. Centrifuge the vial briefly before opening to ensure all lyophilized powder is at the bottom.
2. Reconstitute the protein with sterile distilled water or sterile PBS (phosphate-buffered saline), depending on the product specification. Typical concentrations range from 0.1–0.5 mg/mL for most recombinant proteins, but always check the product datasheet for the recommended concentration.
   • For example: R&D Systems recommends reconstituting at 100 μg/mL in sterile PBS for Angiopoietin-4.
3. Gently mix the solution by swirling or tapping the vial. Do not vortex or pipette vigorously, as this can denature or aggregate the protein.
4. Allow the protein to dissolve at room temperature for 10–15 minutes, occasionally swirling gently.

Preparation for Cell Culture

1. Add a carrier protein (e.g., 0.1% BSA, 5% HSA, or 5% trehalose) to the reconstituted solution if long-term storage is needed or if the protein is carrier-free. This helps stabilize the protein and prevent adsorption to surfaces.
2. Aliquot the solution into small volumes to avoid repeated freeze-thaw cycles.
3. Store aliquots at -20°C to -80°C for long-term storage. For short-term use, keep at 2–8°C for up to 1 week.

Usage in Cell Culture

• Thaw aliquots on ice or at 4°C before use.
• Dilute the protein in cell culture medium to the desired working concentration.
• Filter sterilize (0.22 μm) if necessary before adding to cells.
• Use immediately after dilution for best activity.

Additional Notes

• Always wear appropriate PPE (lab coat, gloves) when handling recombinant proteins.
• Avoid repeated freeze-thaw cycles to maintain protein stability and activity.
• Refer to the specific product datasheet for any unique requirements or recommendations.

By following these steps, you can ensure proper reconstitution and preparation of Recombinant Human Angiopoietin-4 for reliable results in cell culture experiments.