HGF Mouse

What is the molecular structure of mouse HGF and how does it differ from human HGF?

Mouse HGF is a pleiotropic protein belonging to the Plasminogen subfamily of S1 peptidases. It consists of multiple domains with the alpha chain spanning Gln33-Arg495 and the beta chain spanning Val496-Leu728 . Unlike many growth factors, HGF has a complex multidomain structure featuring four kringle structures followed by a serine protease-like domain . While mouse and human HGF share significant homology, species-specific differences exist in binding affinity to the c-MET receptor. Mouse HGF demonstrates high binding affinity to its receptor with an estimated KD<0.2 nM as measured by functional ELISA . When designing cross-species experiments, researchers must account for these structural differences to ensure appropriate interpretation of results.

Where is HGF expressed in normal mouse tissues and what are the baseline concentration ranges?

HGF expression varies significantly across mouse tissues, with important implications for experimental design. In normal mice, serum HGF levels typically range from 5,381-21,185 pg/mL (mean 11,178 pg/mL), while platelet-poor EDTA plasma shows considerably lower levels at 964-6,438 pg/mL (mean 2,809 pg/mL) .

Tissue homogenates reveal distinctive expression patterns:

Cell culture supernatants from tissue homogenates after one day of culture show much lower values: kidney (143 pg/mL), liver (164 pg/mL), and spleen (395 pg/mL) . These baseline values are critical for interpreting changes in experimental conditions and establishing appropriate controls in HGF-focused research.

How does mouse HGF production differ from HGF-like protein production?

Despite structural similarities, mouse HGF and HGF-like protein exhibit fundamentally different cellular origins and expression patterns. HGF is primarily produced by mesenchymal cells including endothelial cells, fibroblasts, and macrophages, acting as a humoral mediator of epithelial-mesenchymal interactions . In contrast, HGF-like protein expression is restricted almost exclusively to hepatocytes, as demonstrated by in situ hybridization analysis .

During embryonic development, this distinction remains evident—at 14 days of gestation, HGF-like protein mRNA is detectable only in hepatocytes, while other developing tissues and even liver hematopoietic cells show no detectable expression . This fundamental difference in cellular origin necessitates careful consideration when designing experiments targeting specific HGF signaling pathways versus those involving HGF-like proteins.

What are the optimal reconstitution and storage conditions for recombinant mouse HGF?

Successful reconstitution and storage of recombinant mouse HGF is critical for maintaining biological activity. For standard preparations containing BSA carrier protein (catalog #2207-HG), reconstitution should be performed at 10 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin . For carrier-free preparations (catalog #2207-HG/CF), a higher concentration of 100 μg/mL in sterile PBS is recommended .

Storage protocols are equally important for experimental reproducibility. Both formulations should be stored in manual defrost freezers, and repeated freeze-thaw cycles must be avoided to maintain protein integrity . For experiments sensitive to carrier proteins, carrier-free preparations should be selected, particularly for applications where BSA might interfere with biological outcomes or detection methods. Proper aliquoting of reconstituted protein is recommended to minimize freeze-thaw cycles across multiple experiments.

What are the most effective techniques for detecting and quantifying HGF in mouse samples?

Quantification of mouse HGF requires careful selection of detection methods based on sample type and expected concentration range. ELISA-based methods provide reliable quantification with sensitivity in the pg/mL range. When using such assays, creating a standard curve with four-parameter logistic (4-PL) curve-fitting is recommended for optimal accuracy .

For tissue homogenate preparation, a standardized protocol involves:

• Rinsing organs with PBS

• Cutting tissues into 1-2 mm pieces

• Homogenizing with a tissue homogenizer in PBS

• Adding an equal volume of cell lysis buffer

• Lysing tissues at room temperature for 30 minutes with gentle agitation

• Removing debris by centrifugation before analysis

When analyzing cell culture supernatants, conditioning periods must be optimized based on cell type—for mouse kidney, liver, and spleen tissues cultured in appropriate media, optimal conditioning periods are typically 18-24 hours . For comparative studies, it's essential to maintain consistent sampling timepoints since HGF secretion can vary significantly over time.

How should mouse HGF activity be measured in functional assays?

Beyond quantification, assessing the biological activity of mouse HGF requires functional assays. A validated approach involves measuring its ability to bind Recombinant Mouse HGF R/c-MET Fc Chimera (e.g., catalog #7065-ME) in a functional ELISA . The EC50 for HGF's biological effects typically ranges from 10-30 ng/mL in most cellular assays .

For in vivo activity assessment, researchers should consider:

• Dose-dependent effects—studies indicate significant biological effects at doses ranging from 50-280 μg/mouse administered over 7-14 days

• Administration timing—both simultaneous and delayed administration protocols can be effective depending on the model

• Delivery method—continuous administration may be required for certain models to maintain therapeutic levels

When evaluating hepatotrophic or renotrophic effects, tissue-specific markers of regeneration should be employed alongside functional assessments of targeted organs to comprehensively characterize HGF activity.

How effective is HGF in preventing bleomycin-induced lung fibrosis in mouse models?

HGF demonstrates significant anti-fibrotic effects in bleomycin-induced lung injury models. Both simultaneous and delayed administration protocols show therapeutic potential, though with different efficacy profiles. In C57BL/6 mice with bleomycin-induced lung injury, low-dose simultaneous HGF administration (50 μg/mouse over 7 days) effectively repressed fibrotic morphological changes at both 2 and 4 weeks post-injury .

Quantitatively, the Ashcroft score (a standardized measure of lung fibrosis) showed significant differences between treated and untreated groups at 4 weeks (3.7 ± 0.4 versus 4.9 ± 0.3, p < 0.05) . Higher dose HGF (280 μg/mouse over 14 days) demonstrated even greater efficacy in both simultaneous and delayed administration protocols, with Ashcroft scores of 2.6 ± 0.4 and 2.4 ± 0.2 versus 4.1 ± 0.2 (p < 0.01) in the control group .

Biochemical analysis of hydroxyproline content, a marker of collagen deposition and fibrosis, further confirmed these findings. Hydroxyproline levels were significantly lower in both simultaneous and delayed HGF administration groups compared to bleomycin-only controls (121.8 ± 8.1% and 113.2 ± 6.2% versus 162.7 ± 4.6%, p < 0.001) . These results indicate that HGF acts as a pulmotrophic factor with therapeutic potential for preventing or treating lung fibrosis in various clinical scenarios.

What are the key experimental considerations when studying HGF's effects on epithelial-mesenchymal interactions in mice?

Investigating HGF's role in epithelial-mesenchymal interactions requires careful experimental design. HGF functions as a humoral mediator of these interactions, exerting multiple effects as a mitogen, motogen, and morphogen on epithelial cells . When designing such studies, researchers should consider:

• Cell-specific HGF production: HGF is primarily produced by mesenchymal cells including endothelial cells, fibroblasts, and macrophages

• Target cell responses: Different epithelial cell populations may respond differently to HGF stimulation

• Dosing regimens: Concentration-dependent effects require dose-response experiments

• Temporal considerations: Both acute and chronic HGF exposure may yield different biological outcomes

• Context dependency: HGF effects may vary significantly in healthy versus injured tissue environments

Advanced co-culture systems incorporating both mesenchymal HGF-producing cells and epithelial target cells can provide more physiologically relevant models than simple HGF supplementation. For in vivo studies, cell-specific knockout or overexpression models targeting either HGF production or c-MET receptor expression in specific cell populations allow for precise dissection of tissue-specific signaling mechanisms.

How do mouse models help differentiate between the roles of HGF and HGF-like protein in liver regeneration?

Mouse models have been instrumental in distinguishing the functions of HGF versus HGF-like protein in liver regeneration. Despite their structural similarities, these proteins exhibit distinctly different expression patterns and likely serve complementary roles in hepatic physiology .

HGF-like protein is expressed almost exclusively by hepatocytes, suggesting an autocrine signaling mechanism within the liver . In contrast, HGF is primarily produced by non-parenchymal liver cells and acts on hepatocytes in a paracrine manner. This fundamental difference suggests potentially distinct roles during liver regeneration.

To effectively differentiate between these roles, researchers can employ:

• Cell-specific conditional knockout models targeting either HGF or HGF-like protein

• Selective neutralizing antibodies that distinguish between the two proteins

• RNA interference approaches targeting the unique regions of each protein's mRNA

• Temporal expression analysis during different phases of liver regeneration

The hepatocyte-specific expression of HGF-like protein observed in both adult mice and 14-day embryos suggests it may play a developmental role distinct from HGF . Comprehensive analysis comparing partial hepatectomy models across these genetic interventions can help elucidate the complementary or potentially redundant functions of these structurally similar yet distinctly expressed growth factors.

How can researchers address variability in baseline HGF levels across different mouse strains?

Significant variability in baseline HGF levels exists across mouse strains, potentially confounding experimental results. Standard reference ranges established in C57BL/6 mice show serum HGF concentrations of 5,381-21,185 pg/mL, but other strains may exhibit different baselines . To address this challenge:

• Perform strain-specific baseline measurements before initiating experiments

• Include strain-matched controls in all experimental designs

• Consider age-matching and sex-matching experimental groups, as these factors can influence HGF levels

• When comparing across strains, normalize experimental changes to strain-specific baselines rather than absolute values

• Report relative changes (fold-change) alongside absolute concentrations

For longitudinal studies, establish individual baseline measurements where possible, as intra-strain variability can also be substantial. The wide reference range even within standardized strains (SD of 3,678 pg/mL in serum samples) highlights the importance of adequate sample sizes for statistical power .

What strategies can prevent the loss of HGF activity during experimental manipulations?

Maintaining HGF biological activity throughout experimental procedures requires specific technical considerations. To preserve activity:

• Minimize freeze-thaw cycles by preparing appropriately sized single-use aliquots

• For carrier-containing preparations, reconstitute at 10 μg/mL in sterile PBS with 0.1% albumin

• For carrier-free preparations, use higher concentrations (100 μg/mL) to minimize surface adsorption

• Store reconstituted protein at recommended temperatures and avoid repeated temperature cycling

• Use low-binding tubes and pipette tips for handling dilute solutions

• Consider addition of stabilizing agents for long-term storage

• Validate activity periodically using functional binding assays

When designing in vivo delivery systems, consider that continuous administration approaches may better maintain therapeutic levels compared to bolus injections . For extended release applications, osmotic pumps or engineered matrices that gradually release HGF can help maintain consistent biological activity throughout the experimental period.

How should researchers interpret conflicting data between HGF protein levels and biological activity?

Discrepancies between measured HGF protein concentrations and observed biological activity frequently challenge researchers. Several factors can contribute to such conflicts:

• Post-translational modifications affecting activity but not detection by standard ELISAs

• Presence of endogenous inhibitors in biological samples

• Matrix effects in complex samples interfering with accurate quantification

• Partial proteolysis generating immunoreactive fragments without biological activity

• Variations in c-MET receptor expression or signaling in target cells

When facing such conflicts, a multi-faceted approach is recommended:

• Compare results from multiple detection methods (ELISA, Western blot, mass spectrometry)

• Assess both total and active HGF using functional binding assays

• Evaluate downstream signaling markers (phosphorylated c-MET, ERK, AKT) as proxies for activity

• Consider dose-response studies with recombinant protein as positive controls

• Examine potential presence of inhibitors through mixing experiments with known active HGF

Remember that the biological context may dramatically alter HGF activity—interactions with extracellular matrix components, co-receptors, or competing ligands can all modulate effective signaling despite consistent protein levels.

What emerging technologies are advancing our understanding of HGF signaling in mouse models?

Recent technological advances offer unprecedented insights into HGF signaling dynamics in mouse models. These emerging approaches include:

• Single-cell RNA sequencing to identify cell-specific responses to HGF stimulation

• CRISPR/Cas9-mediated genome editing for precise manipulation of HGF signaling components

• Tissue-specific inducible expression systems allowing temporal control of HGF production

• Intravital microscopy techniques for real-time visualization of HGF-induced cell behavior

• Phosphoproteomic analysis to comprehensively map HGF-activated signaling networks

• Organoid culture systems providing three-dimensional contexts for studying HGF biology

These technologies enable more sophisticated experimental designs that can address longstanding questions about context-dependent HGF functions. For example, combining single-cell analysis with spatial transcriptomics can reveal how HGF gradient formation affects cell-specific responses within complex tissues—information critical for understanding HGF's role in development, regeneration, and disease.

How might comparative studies between mouse HGF and HGF-like protein advance therapeutic applications?

Comparative analysis of mouse HGF and HGF-like protein offers promising avenues for therapeutic development. Despite sharing structural domains including four kringle structures and serine protease-like regions, these proteins exhibit distinctly different expression patterns—HGF is produced by mesenchymal cells while HGF-like protein is specifically expressed in hepatocytes .

This fundamental difference suggests potential complementary or specialized functions that might be therapeutically exploitable. Research directions might include:

• Investigating whether HGF-like protein could provide hepatocyte-specific effects with fewer systemic side effects than HGF

• Determining whether structural differences confer distinct receptor binding or signaling properties

• Exploring potential synergistic or antagonistic interactions between the two proteins

• Developing selective agonists or antagonists based on unique structural features

• Examining differential regulation under pathological conditions

Understanding the evolutionary conservation and divergence between these related proteins across species could provide further insights into their specialized functions. Such knowledge could ultimately lead to more targeted therapeutic approaches for tissue-specific regeneration or fibrosis treatment.

What are the most promising translational applications of mouse HGF research for human disease?

Mouse HGF research has illuminated several promising translational pathways for human disease intervention. The demonstrated efficacy of both simultaneous and delayed HGF administration in bleomycin-induced lung fibrosis models suggests therapeutic potential for conditions characterized by pulmonary fibrosis . In these models, HGF treatment significantly reduced fibrosis as measured by both histological scoring and biochemical analysis of collagen deposition .

Additional promising translational directions include:

• Hepatic regeneration therapies—building on HGF's established role as a potent hepatotrophic factor

• Renal protective strategies—leveraging HGF's renotrophic properties in acute and chronic kidney injury

• Tissue engineering applications—using HGF to promote epithelial morphogenesis and vascularization

• Cancer therapeutics—developing approaches targeting dysregulated HGF/c-MET signaling in tumors

• Fibrosis treatment beyond the lung—exploring anti-fibrotic effects in liver, kidney, and other organs

Translation to human applications requires addressing scale-up challenges, optimizing delivery systems, and developing human-specific reagents. Comparative studies between mouse and human HGF signaling are essential, as species-specific differences in receptor binding, downstream signaling, and physiological responses must be carefully characterized before clinical translation.