Recombinant Human Hepatocyte growth factor (HGF) (Active)

What is recombinant human HGF and how is it structurally characterized?

Recombinant human HGF is a protein produced using various expression systems to mimic the biological activity of native human HGF. Structurally, commercial rh-HGF typically consists of amino acids Gln36-Ser655 with potential modifications such as C-terminal tags (e.g., 10-His tag) to facilitate purification . High-quality rh-HGF preparations typically achieve >98% purity as assessed by SDS-PAGE and HPLC . Expression systems commonly include baculovirus-infected insect cells, which help ensure proper post-translational modifications essential for biological activity .

What are the optimal storage conditions for maintaining rh-HGF stability?

For optimal stability, rh-HGF should be stored in a manual defrost freezer with repeated freeze-thaw cycles strictly avoided . The protein is typically supplied as a 0.2 μm filtered solution in buffer containing Tris, NaCl, and CaCl₂ . Upon receipt, the protein should be immediately stored at the recommended temperature. For carrier-free formulations (those without BSA stabilizer), extra precautions may be necessary as these preparations can be less stable at dilute concentrations. Some preparations may require storage in small aliquots to minimize stability issues from repeated freeze-thaw cycles.

What are the primary biological functions of HGF relevant to research applications?

HGF demonstrates diverse biological functions critical to multiple physiological processes. In embryonic development, HGF and its receptor c-Met are essential, as targeted destruction of either leads to embryonic lethality with specific damage to liver and placental development . In adult tissues, HGF functions as a potent mitogen for hepatocytes, stimulating cell proliferation during liver regeneration . It also acts as an anti-apoptotic factor, significantly inhibiting hepatocyte death while stabilizing structural and vascular integrity in acute liver failure models . Additionally, HGF signaling through the c-Met receptor plays crucial roles in regulating cell migration, morphogenesis, and tissue repair mechanisms in various organ systems.

How can researchers validate the biological activity of rh-HGF preparations?

Activity validation of rh-HGF requires specific functional assays. A standardized approach involves measuring enzymatic activity using fluorogenic peptide substrates. Following the established protocol, rh-HGF Activator is first activated by incubation with bacterial Thermolysin at 37°C, followed by inactivation of Thermolysin using 1,10-Phenanthroline . The activated rh-HGF can then be assessed using substrate MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂, with fluorescence measured at excitation/emission wavelengths of 320/405 nm . Specific activity calculation follows:

$\text{Specific Activity (pmol/min/μg)} = \frac{\text{Measured RFU/min} \times \text{Conversion Factor}}{\text{Amount of Enzyme (μg)}}$

For validated rh-HGF preparations, the specific activity should exceed 15 pmol/min/μg under the described conditions .

What are the optimal dosing strategies for rh-HGF in preclinical liver failure models?

Based on clinical protocols, initial dosing of rh-HGF should be carefully titrated. For preclinical models of acute liver failure, researchers should consider the following evidence-based approach:

• Starting dose: Begin with 0.6 mg/m² as established in human clinical trials

• Delivery method: Administer intravenously with a stepwise increase over 3 hours to minimize potential hypotensive effects

• Duration: Treatment regimens of up to 14 days have been clinically tested, with subsequent 14-day observation periods

• Dose escalation: For dose-response studies, evaluate escalating doses (0.6, 1.2, 1.8, and 2.4 mg/m²) with appropriate safety monitoring

When designing dosing studies, researchers should monitor hepatic function parameters and vital signs, particularly blood pressure, as hypotensive effects may occur during administration.

How does the HGF/c-Met signaling axis interact with other pathways in liver regeneration models?

The HGF/c-Met signaling axis demonstrates complex interactions with multiple regulatory pathways during liver regeneration. The activation system involves urokinase-type plasminogen activator (uPA), which facilitates release of preformed HGF from the extracellular matrix . This process is dynamically regulated in several ways:

• Toll-like receptor 3 (TLR3) upregulates uPA, increasing HGF availability during liver regeneration

• Nuclear factor ImerC (NFI-C) deletion leads to overexpression of plasminogen activator inhibitor 1 (PAI-1), inhibiting uPA activity and HGF signaling

• Peroxisome proliferator-activated receptor gamma (PPARγ) inhibits liver regeneration by suppressing HGF/c-Met/ERK1/2 pathway activation

• Bone morphogenetic protein 9 (BMP9) establishes signal crossover with HGF/c-Met through ALK1, modulating SMAD1 (pro-survival) and p38MAPK (pro-apoptotic) pathways

Understanding these interactions is critical when designing experiments targeting specific aspects of the regenerative response.

What controls should be included when studying rh-HGF effects in cell culture models?

For rigorous experimental design when studying rh-HGF effects, include the following controls:

• Negative controls:

  • Vehicle control (buffer identical to rh-HGF formulation buffer)

  • Heat-inactivated rh-HGF (to control for non-specific protein effects)

  • Untreated cells (baseline comparison)

• Positive controls:

  • Known HGF-responsive cell line (e.g., HepG2 for hepatocyte studies)

  • Established dose of epidermal growth factor (EGF) that produces comparable proliferative effects

• Pathway validation controls:

  • c-Met inhibitor (e.g., SGX523) to confirm specificity of observed effects

  • ERK1/2 inhibitors to assess downstream signaling dependency

• Additional controls for carrier-free preparations:

  • Parallel experiments with BSA-containing and carrier-free preparations to identify potential carrier effects

How should researchers prepare and administer rh-HGF for in vivo acute liver failure studies?

Based on clinical protocols, researchers should consider the following standardized approach for in vivo studies:

• Initial preparation:

  • Dilute rh-HGF in appropriate vehicle (typically Tris buffer with NaCl and CaCl₂)

  • Filter solution through a 0.2 μm filter under sterile conditions

  • Prepare fresh immediately before administration

• Administration protocol:

  • For intravenous administration, use a stepwise infusion approach (10% of total dose in first hour, 30% in second hour, 60% in third hour) to minimize potential hypotensive effects

  • Monitor vital signs during administration, particularly blood pressure

• Exclusion criteria for experimental animals:

  • Evidence of renal involvement (proteinuria, elevated creatinine)

  • Pre-existing major cardiovascular abnormalities

  • Severe comorbidities that may confound results

This approach mirrors clinical trial protocols and helps ensure translational relevance of findings.

What biomarkers should be monitored to assess rh-HGF efficacy in liver injury models?

To comprehensively evaluate rh-HGF efficacy in liver injury models, researchers should monitor multiple categories of biomarkers:

• Standard liver function parameters:

  • Serum transaminases (ALT, AST)

  • Bilirubin (total and direct)

  • Albumin and total protein

  • Prothrombin time/INR

• Specific markers of hepatocyte proliferation:

  • Ki-67 or PCNA immunostaining

  • BrdU incorporation

  • Cyclin expression profiles

• Apoptosis markers:

  • TUNEL staining

  • Caspase-3/7 activity

  • Bcl-2/Bax ratio

• HGF/c-Met pathway activation markers:

  • Phosphorylated c-Met levels

  • Phosphorylated ERK1/2 levels

  • Downstream targets like cyclin D1

• Inflammatory and stress markers:

  • Proinflammatory cytokines (IL-6, TNF-α)

  • Oxidative stress markers

This multi-parameter approach provides a comprehensive assessment of therapeutic efficacy.

How can researchers distinguish between exogenous rh-HGF and endogenous HGF in experimental systems?

Distinguishing between exogenous rh-HGF and endogenous HGF is methodologically challenging but can be approached through several strategies:

• Tagged rh-HGF:

  • Utilize commercially available rh-HGF with specific tags (e.g., His-tag)

  • Detect using tag-specific antibodies via Western blot or immunoprecipitation

• Pharmacokinetic approach:

  • Establish baseline endogenous HGF levels before administration

  • Monitor rapid changes in total HGF levels at early timepoints after administration

  • Analyze plasma half-life and clearance patterns that differ between endogenous and recombinant proteins

• Species-specific detection:

  • In xenograft models, use species-specific anti-HGF antibodies that distinguish human rh-HGF from host (e.g., mouse) HGF

• Mass spectrometry analysis:

  • Identify unique peptide signatures that distinguish recombinant from endogenous HGF

  • Particularly useful when recombinant protein contains sequence modifications

What are the critical considerations when transitioning from in vitro to in vivo rh-HGF studies?

Researchers transitioning from in vitro to in vivo studies should address several key factors:

• Dosage translation:

  • In vitro effective concentrations cannot be directly translated to in vivo dosing

  • Consider allometric scaling based on body surface area

  • Begin with established safe doses (e.g., 0.6 mg/m²) and adjust based on pilot study results

• Delivery considerations:

  • Account for first-pass metabolism and bioavailability

  • Consider the pharmacokinetic profile of rh-HGF in the target species

  • Implement stepwise infusion protocols to minimize potential hypotensive effects

• Model selection:

  • Ensure the liver injury model appropriately activates the HGF/c-Met pathway

  • Consider genetic background effects on response to rh-HGF

  • Validate c-Met receptor expression in the model system

• Formulation adjustments:

  • For in vivo studies, carrier-free rh-HGF preparations may be preferable to avoid potential BSA-related effects

  • Ensure buffer compatibility with in vivo administration

• Timing considerations:

  • Determine optimal intervention window based on model characteristics

  • Consider repeated dosing strategies (up to 14 days as in clinical protocols)

How can researchers assess and optimize the activation state of rh-HGF preparations?

HGF is produced as a single-chain precursor that requires proteolytic cleavage for full biological activity. Researchers can assess and optimize activation through these approaches:

• Proteolytic activation:

  • Incubate rh-HGF with specific proteases like Thermolysin (4.3 μg/mL) in activation buffer containing calcium at 37°C for 1 hour

  • Stop activation with metalloprotease inhibitors like 1,10-Phenanthroline (10 mM final concentration)

• Verification methods:

  • Western blot analysis to confirm conversion from single-chain to two-chain form

  • Functional assays using fluorogenic peptide substrates with excitation/emission at 320/405 nm

  • Calculate specific activity (target >15 pmol/min/μg)

• Optimization strategies:

  • Adjust protease concentration and incubation time based on batch-specific requirements

  • Include dextran sulfate (10 μg/mL) in activation buffer to enhance proteolytic efficiency

  • Test multiple activation conditions in pilot experiments to identify optimal parameters

What are common issues affecting reproducibility in rh-HGF experiments and how can they be addressed?

Several factors can impact reproducibility in rh-HGF experiments:

• Protein stability issues:

  • Avoid repeated freeze-thaw cycles

  • Store in small single-use aliquots

  • Use carrier-free preparations only when necessary for specific applications

• Variability in activation:

  • Standardize activation protocol with precise timing and temperature control

  • Verify activation before each experiment using activity assays

  • Consider batch testing and normalization based on specific activity rather than protein concentration

• Cell responsiveness variability:

  • Maintain consistent cell passage number across experiments

  • Verify c-Met receptor expression levels

  • Standardize cell density and culture conditions

• HGF/c-Met pathway modulation by experimental conditions:

  • Control for hypoxia, which can decrease HGF expression

  • Account for effects of culture media components on pathway activity

  • Consider potential crosstalk with other signaling pathways (e.g., PPARγ)

How should researchers interpret conflicting data regarding rh-HGF efficacy across different model systems?

When facing conflicting data about rh-HGF efficacy across different models, consider these analytical approaches:

• Model-specific variables:

  • Compare extent of liver injury/regenerative stimulus between models

  • Assess baseline c-Met expression and activation state

  • Evaluate potential differences in downstream pathway components

• Treatment protocol differences:

  • Analyze timing of rh-HGF administration relative to injury

  • Compare dosing strategies and administration routes

  • Evaluate duration of treatment and assessment timepoints

• Strain/species differences:

  • Consider genetic background effects on regenerative capacity

  • Assess species-specific differences in HGF/c-Met binding affinity

  • Analyze variation in proteolytic activation mechanisms

• Contextual factors:

  • Evaluate influence of inflammatory milieu on response to rh-HGF

  • Consider interactions with other growth factors and cytokines

  • Assess metabolic state of target tissue (e.g., hypoxia significantly affects HGF expression)

A systematic analysis of these factors can help reconcile apparently conflicting results and identify the specific conditions under which rh-HGF demonstrates efficacy.