HGF Human, HEK

What is HGF and what cellular receptors does it interact with?

Hepatocyte Growth Factor (HGF) is a growth factor secreted primarily by cells of mesenchymal origin that binds to the c-Met receptor on endothelial cells. The binding of HGF to c-Met initiates intracellular signaling cascades that regulate various cellular processes including proliferation, migration, and survival. HGF not only stimulates endothelial cell growth without inducing vascular smooth muscle cell proliferation but also accelerates re-endothelialization while causing minimal intimal hyperplasia . This selective activity makes HGF particularly valuable in potential therapeutic applications for vascular diseases. The HGF-c-Met interaction can be detected and quantified in cell and tissue samples using techniques such as antibody proximity assays, which provide more precise measurements than traditional immunohistochemistry approaches .

What are the key biological functions of HGF in human systems?

HGF serves multiple critical functions in human physiology. It prevents endothelial cell death through anti-apoptotic activities and represents one of the major determinants in regulating epithelial cell state transitions between quiescence and proliferation during development and tissue repair . In pathological conditions such as burn injuries, HGF works through distinct pathways to modulate hepatic acute phase reactant proteins and cytokine expression . When administered in combination with recombinant human growth hormone (rhGH) in burn models, HGF demonstrates additive effects on constitutive hepatic proteins and partial inhibitory effects on acute phase protein and cytokine expression, while exerting strong mitogenic effects on hepatocytes . Additionally, HGF has significant angiogenic properties, making it valuable for research in ischemic conditions when co-administered with other factors like VEGF165 .

What challenges exist regarding the in vivo stability of HGF?

A significant challenge in HGF research and therapeutic application is its very short half-life of less than 3-5 minutes in vivo . This brief biological half-life severely limits the therapeutic potential of direct HGF administration. Furthermore, while endogenous HGF levels increase after tissue injury, they rarely reach sufficient concentrations for complete tissue repair due to this rapid clearance . Researchers have developed several strategies to overcome this limitation, including genome editing techniques to create stable expression systems. One approach involves integrating a Dox-inducible HGF-expression system into the PPP1R12C site (safe harbor site) on human chromosome 19 using transcription activator-like effector nucleases (TALEN)-mediated genome editing, enabling long-term and controllable HGF secretion . These controlled expression systems aim to maintain therapeutic HGF concentrations while avoiding potential oncogenic effects from unregulated HGF-Met signaling.

How can HEK293 cells be optimized for HGF expression?

HEK293 cells represent an excellent platform for HGF expression due to their high transfection efficiency and robust protein production capabilities. When developing HGF expression systems in HEK293 cells, researchers should consider:

• Promoter selection: The EF1α promoter has demonstrated effectiveness for driving expression elements like rtTA in tetracycline-inducible systems for HGF .

• Inducible systems: Tetracycline/doxycycline (TetOn) inducible systems allow for controlled HGF expression, which is crucial since constitutive HGF-Met signaling may trigger tumor growth .

• Optimization of doxycycline concentration: Experimental data suggests that 5-7 μg/ml of doxycycline provides optimal HGF secretion in transfected human cells .

• Transfection method: HEK293 cells demonstrate transfection efficiencies exceeding 50% with standard plasmid vectors, significantly higher than other cell types like adipose-derived stem cells (~10%), making them particularly suitable for HGF expression studies .

• Validation approaches: Following transfection and selection, validate stable HGF-expressing HEK293 cell lines using techniques such as ELISA for secreted HGF and immunoblotting for intracellular HGF expression.

What methods are available for detecting and quantifying HGF in experimental samples?

Several methods have been developed for reliable detection and quantification of HGF in research samples:

• Antibody Proximity Assays: These assays can detect HGF in formalin-fixed, paraffin-embedded (FFPE) specimens with high specificity. The technique involves using a goat polyclonal anti-human HGF antibody and a mouse monoclonal anti-human HGF antibody (clone SBF5) in a two-antibody format . This approach demonstrates approximately a 3-log10 dynamic range when comparing HGF expression between different cell lines .

• ELISA: Standard ELISA techniques correlate well with proximity assay measurements in cell lysates, providing a complementary approach for HGF quantification .

• HGF/c-MET Complex Detection: Specialized antibody proximity assays can detect not just HGF levels but also HGF/c-MET complexes, providing insights into active signaling rather than just protein expression. This technique employs rabbit anti-MET antibody (clone SP44) together with human anti-HGF antibody conjugated to a VeraTag reporter .

• Validation Controls: To ensure specificity, assays should incorporate appropriate controls, including isotype antibody-matched controls (which typically yield signals less than 5-20% of specific assay signals) and neutralizing antibody controls to confirm specificity of detection .

How can researchers establish an inducible HGF expression system?

To establish an inducible HGF expression system, researchers can follow this methodological approach:

• Construct Design: Create a TetOn inducible system where HGF expression is controlled by tetracycline/doxycycline treatment. This involves:

  • Cloning human HGF cDNA into an appropriate vector

  • Incorporating the TetOn regulatory elements

  • Including a constitutive promoter (e.g., EF1α) to drive the reverse tetracycline transactivator (rtTA) expression

• Safe Harbor Integration: For stable long-term expression, integrate the construct into the PPP1R12C site on human chromosome 19 (a known safe harbor site) using genome editing techniques like TALEN-mediated approaches .

• Optimal Induction Conditions: Determine the optimal doxycycline concentration for HGF induction, which is typically in the range of 5-7 μg/ml for human cells .

• Verification: Confirm the functionality of the inducible system by:

  • Measuring HGF secretion in conditioned media with and without doxycycline treatment

  • Verifying that HGF levels increase proportionally with doxycycline concentration within the effective range

  • Confirming that HGF expression ceases when doxycycline is removed

• Characterization: Ensure that the modified cells maintain their original phenotypic characteristics, particularly when working with stem cells, by assessing standard cell-type-specific markers before and after doxycycline treatment .

How does HGF function in combination therapy approaches?

HGF demonstrates significant therapeutic potential when used in combination with other growth factors or therapeutic agents:

• HGF with Growth Hormone: The combination of recombinant human growth hormone (rhGH) and HGF has shown synergistic effects in burn injury models. In experimental studies using Sprague-Dawley rats with 60% TBSA third-degree scald burns, administration of rhGH (2.5 mg/kg/day subcutaneously) plus HGF (200 μg/kg intravenously every 12 hours) resulted in:

  • Increased serum albumin levels on days 5 and 7 post-burn

  • Elevated transferrin levels on day 7 post-burn

  • Decreased haptoglobin (an acute phase protein) on day 5 post-burn

  • Enhanced liver total protein content

  • Increased hepatocyte proliferation on days 1, 2, 5, and 7 post-burn

These findings suggest that the rhGH/HGF combination exerts additive effects on constitutive hepatic proteins while partially inhibiting acute phase protein and cytokine expression .

• HGF with VEGF165: Co-transfer of HGF and vascular endothelial growth factor 165 (VEGF165) genes has demonstrated robust angiogenic effects in ischemic skeletal muscle. This combination approach leverages complementary mechanisms, with HGF and VEGF165 potentially activating different aspects of the angiogenic process, resulting in enhanced therapeutic outcomes compared to single-gene approaches .

• Signaling Pathway Considerations: When designing combination approaches, researchers should consider potential pathway interactions. For example, the ERK1/2 pathway has been implicated in the mechanism of combined HGF and VEGF165 administration effects in human umbilical vein endothelial cells (HUVECs) .

What are the considerations for HGF gene therapy approaches?

When developing HGF gene therapy approaches, researchers should consider several critical factors:

• Vector Selection: Novel cytomegalovirus-based (CMV) plasmid vectors with codon-optimized human HGF genes have demonstrated efficacy in experimental models . The vector design significantly impacts transfection efficiency and expression duration.

• Delivery Method: For skeletal muscle applications, intramuscular low-voltage electroporation has shown effectiveness for plasmid-based gene transfer . The delivery method must be optimized for the specific target tissue and desired expression profile.

• Expression Validation: Multiple approaches should be used to confirm functional expression:

  • ELISA of tissue homogenates to quantify HGF protein levels

  • Functional activity assays, such as tube formation in human umbilical vein endothelial cell (HUVEC) cultures, to verify biological activity of the secreted HGF

  • In vivo validation in appropriate disease models, such as limb ischemia models for angiogenic applications

• Controlled Expression Systems: Given that continuous HGF-Met signaling can potentially trigger tumor growth, incorporating inducible expression systems is crucial for safety in gene therapy applications . The TetOn inducible system allows for regulated HGF expression through doxycycline administration.

• Combined Approaches: Consider co-administration with complementary factors, as combined gene transfer may provide enhanced therapeutic effects compared to single-gene approaches .

How can researchers investigate HGF/c-MET signaling complexes?

Investigating HGF/c-MET signaling complexes requires specialized techniques beyond simple protein expression analysis. Researchers can employ the following methodological approach:

• Antibody Proximity Assays: These assays can detect and quantify HGF/c-MET complexes in FFPE specimens through a process involving:

  • Incubation with rabbit anti-MET antibody (clone SP44) together with human anti-HGF antibody conjugated to a VeraTag reporter

  • Addition of biotin-conjugated goat anti-rabbit antibody

  • Addition of streptavidin-methylene blue photosensitizer

  • Illumination of the sample to release VeraTag reporters in close proximity

• Validation Controls: Include appropriate controls to ensure specificity:

  • Isotype controls (typically yielding signals less than 20% of specific signals)

  • HGF neutralizing antibody controls to demonstrate specificity of detected complexes

  • Concentration gradients to establish dose-response relationships

• Correlation with Phosphorylation: Correlate HGF/c-MET complex formation with downstream signaling events by measuring c-MET phosphorylation status in parallel samples .

• In Vitro Models: Establish model systems using cell lines with known HGF/c-MET characteristics. For example:

  • Use unstimulated and HGF-stimulated A549 cells as a controlled model system

  • Employ cell lines with autocrine HGF/c-MET signaling, such as certain glioma cell lines (U118, Ln18, and U87MG), which exhibit 12-40 fold higher levels of HGF than other cell lines like U138

• Neutralizing Antibody Studies: Pre-incubate HGF with neutralizing antibodies (e.g., clone 24612) to demonstrate specificity of the detected complex and confirm functional relevance through corresponding decreased c-MET phosphorylation .

What factors affect HGF expression and activity in experimental systems?

Several factors can significantly impact HGF expression and activity in research settings:

• Cell Type Selection: Different cell types demonstrate varying capacities for HGF expression and transfection efficiency. Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) show higher transfection efficiency (>50%) compared to adipose-derived stem cells (ADSCs, ~10%), resulting in significantly different HGF expression levels even with identical constructs .

• Expression System Stability: The integration site of HGF expression constructs affects long-term stability. Integration into safe harbor sites like PPP1R12C on chromosome 19 provides more consistent expression compared to random integration approaches .

• Induction Parameters: For inducible systems, doxycycline concentration significantly impacts HGF expression levels. Experimental data indicates optimal induction occurs at 5-7 μg/ml of doxycycline, with concentrations outside this range potentially resulting in suboptimal expression .

• Detection Method Sensitivity: Different detection methods have varying sensitivities and dynamic ranges. Antibody proximity assays demonstrate approximately a 3-log10 dynamic range, allowing for detection of a wide range of HGF expression levels .

• Autocrine Signaling: Some cell lines exhibit autocrine HGF/c-MET signaling, which can confound experimental results. Glioma cell lines U118, Ln18, and U87MG naturally secrete high levels of HGF and may have activated HGF/c-MET pathways independent of exogenous HGF administration .

How can researchers address the short half-life of HGF in experimental and therapeutic applications?

The extremely short half-life of HGF (<3-5 minutes in vivo) presents a significant challenge for both research and therapeutic applications . Researchers have developed several strategies to address this limitation:

• Gene Therapy Approaches: Rather than administering HGF protein directly, gene therapy enables sustained local production of HGF. This can be accomplished using:

  • Plasmid vectors with CMV-based promoters for transient expression

  • Stable genomic integration using TALEN-mediated approaches for long-term expression

• Inducible Expression Systems: TetOn inducible systems allow for controlled expression of HGF, enabling researchers to maintain therapeutic levels while avoiding potential negative effects of constitutive HGF-Met signaling .

• Combination Therapies: Co-administration of HGF with other factors, such as growth hormone or VEGF165, can enhance therapeutic effects through complementary mechanisms, potentially requiring lower effective doses of HGF .

• Cell-Based Delivery Systems: Genetically modified cells, particularly stem cells like hUCB-MSCs, can serve as delivery vehicles that continuously produce HGF in the target tissue over extended periods .

• Protein Engineering: Although not mentioned in the provided search results, protein engineering approaches to increase HGF stability through modifications of the amino acid sequence or conjugation with stabilizing moieties represent another potential strategy being explored in the field.

What innovative approaches are being developed for controlled HGF expression?

Recent advances in controlled HGF expression include:

• Genome Editing for Safe Harbor Integration: TALEN-mediated genome editing techniques enable precise integration of HGF expression systems into safe harbor sites like PPP1R12C on chromosome 19. This approach provides several advantages:

  • Long-term stable expression

  • Reduced risk of insertional mutagenesis

  • Maintenance of normal cellular phenotype and function

• Advanced Inducible Systems: The TetOn inducible system allows for temporal control of HGF expression through administration of doxycycline. This system comprises:

  • EF1α promoter driving rtTA expression

  • Tetracycline response element controlling HGF transcription

  • Precise dose-dependent regulation of HGF secretion

• Cell-Type Optimization: Research has identified optimal cell types for HGF expression, with hUCB-MSCs demonstrating superior characteristics:

  • High transfection efficiency (>50%)

  • Low immunogenicity

  • Maintained stemness properties with or without doxycycline treatment

  • Effective secretion of functional HGF

• Combined Gene Transfer Systems: Novel vectors enabling co-expression of multiple therapeutic genes, such as HGF and VEGF165, represent an emerging approach for enhanced therapeutic effects through complementary mechanisms of action .

How is HGF research advancing our understanding of disease mechanisms?

HGF research has provided significant insights into various disease mechanisms:

• Vascular Diseases: Studies on HGF's effects on endothelial cells have advanced our understanding of angiogenesis and vascular repair mechanisms. HGF stimulates endothelial cell growth without inducing vascular smooth muscle cell proliferation, accelerates re-endothelialization, and causes minimal intimal hyperplasia, revealing its unique role in vascular homeostasis .

• Burn Injury Recovery: Research on combined rhGH and HGF administration in burn models has elucidated the complex interplay between growth factors, acute phase responses, and tissue regeneration. The combination therapy increased serum albumin and transferrin levels while decreasing haptoglobin, demonstrating differential effects on constitutive proteins versus acute phase reactants .

• Cancer Biology: Investigation of HGF/c-MET signaling complexes in tumors has enhanced our understanding of autocrine signaling mechanisms in cancer progression. Studies have shown that certain glioma cell lines (U118, Ln18, U87MG) exhibit high levels of HGF and possess an HGF/c-MET autocrine loop, which may contribute to their malignant phenotype .

• Ischemic Conditions: Research on combined VEGF165 and HGF gene transfer has provided insights into the complementary mechanisms of angiogenic factors in ischemic tissues. These studies suggest potential synergistic effects through activation of overlapping yet distinct signaling pathways, such as the ERK1/2 pathway in endothelial cells .

What are the future directions for HGF therapeutic applications?

Future directions for HGF therapeutic applications include:

• Refined Gene Therapy Approaches: Development of improved vectors and delivery systems for HGF gene therapy, focusing on:

  • Enhanced tissue specificity through targeted promoters

  • Optimized codon usage for increased expression efficiency

  • Novel delivery methods beyond electroporation, potentially including nanoparticle-based approaches

• Combination Therapies: Further exploration of synergistic combinations with other growth factors or therapeutic agents:

  • HGF with VEGF family members for enhanced angiogenesis

  • HGF with anti-inflammatory agents for comprehensive tissue repair

  • HGF with cell-based therapies for integrated regenerative approaches

• Personalized Medicine Applications: Development of patient-specific approaches based on HGF/c-MET pathway status:

  • Diagnostic tools to identify patients who would benefit most from HGF therapy

  • Tailored dosing regimens based on individual response markers

  • Combination approaches specific to patient disease characteristics

• Advanced Regulatory Systems: Further refinement of inducible expression systems to achieve:

  • More precise control over HGF expression levels

  • Reduced "leakiness" in the absence of inducer

  • Tissue-specific induction capabilities

  • Response to endogenous disease-related signals rather than exogenous inducers

• Expanded Disease Applications: Extension of HGF therapeutic approaches to additional conditions beyond current research focus areas, potentially including chronic liver diseases, kidney disorders, neurodegenerative conditions, and various ischemic pathologies .

What experimental models are most appropriate for studying HGF functions?

The selection of appropriate experimental models is crucial for studying specific aspects of HGF biology:

• Cell Line Models:

  • HEK293 cells: Excellent for HGF expression studies due to high transfection efficiency (>50%)

  • A549 cells: Suitable for studying exogenous HGF stimulation effects as they exhibit low baseline HGF expression

  • Glioma cell lines (U118, Ln18, U87MG): Valuable for studying autocrine HGF/c-MET signaling as they naturally secrete HGF

  • HUVECs: Appropriate for functional assays of HGF activity, such as tube formation assays

• In Vivo Models:

  • Mouse hindlimb ischemia model: Effective for studying HGF's angiogenic effects in skeletal muscle

  • Rat burn injury model: Suitable for investigating HGF's role in acute phase responses and tissue repair following thermal injury

• Ex Vivo Systems:

  • Murine skeletal muscle explants: Useful for studying HGF effects in a tissue context while maintaining normal cellular architecture

  • FFPE tissue samples: Valuable for developing and validating detection methods for HGF and HGF/c-MET complexes in human tissues

• Model Selection Considerations:

  • Research question specificity (e.g., angiogenesis vs. tissue repair)

  • Required detection sensitivity and dynamic range

  • Need for human vs. animal systems

  • Acute vs. chronic effects of HGF

How should researchers validate HGF activity in experimental systems?

Comprehensive validation of HGF activity requires multiple complementary approaches:

• Protein Expression Validation:

  • ELISA of conditioned media or tissue homogenates to quantify HGF protein levels

  • Immunohistochemistry to visualize HGF distribution in tissues

  • Antibody proximity assays for sensitive detection in FFPE samples

• Receptor Engagement Verification:

  • HGF/c-MET complex detection using specialized antibody proximity assays

  • Phosphorylation status of c-MET receptor as evidence of functional engagement

  • Competition studies with HGF neutralizing antibodies to confirm specificity

• Functional Activity Assessment:

  • Endothelial tube formation assays using HUVECs to evaluate angiogenic potential

  • Cell proliferation assays to measure mitogenic effects

  • Migration assays to assess motogenic properties

  • Anti-apoptotic activity measurement in relevant cell types

• In Vivo Functional Validation:

  • Tissue perfusion measurements in ischemia models

  • Histological assessment of angiogenesis (capillary density)

  • Protein expression analysis in target tissues

  • Physiological outcome measures specific to the disease model

• Control Experiments:

  • Isotype antibody controls for detection specificity

  • Dose-response relationships to establish physiological relevance

  • Comparison with established HGF sources or standards

  • Inhibition studies using pathway-specific inhibitors to confirm mechanism