PLGF2 Human, HEK

What is the molecular structure of human PLGF2 protein expressed in HEK293 cells?

Human PLGF2 protein expressed in HEK293 cells contains amino acids Leu 19 to Arg 170 (Accession # NP_002623.2). When tagged with C-terminal polyhistidine, it has a calculated molecular weight of 18.2 kDa but migrates as 28-33 kDa under reducing conditions in SDS-PAGE due to glycosylation. The protein is typically stored in 100 mM Acetic Acid buffer at pH 3.0 and maintained at -20°C for optimal stability. This recombinant protein system allows for proper post-translational modifications that are critical for its biological function .

How does PLGF2 differ from other PLGF isoforms in structure and function?

PLGF2 differs from other PLGF isoforms, particularly PLGF1, in several critical ways. The main functional distinction is PLGF2's ability to bind heparin and interact with neuropilin receptors (NRP1/NRP2) in a heparin-dependent manner, while PLGF1 binds only to VEGFR-1 (Flt-1). This additional binding capability gives PLGF2 distinct biological properties. In humans, four PLGF isoforms (PLGF1-4) are generated through alternative splicing, whereas mice express only a single PLGF form analogous to human PLGF2 . These structural differences significantly impact their biological activities, as PLGF2 shows broader receptor interactions that influence angiogenic responses and cellular recruitment patterns .

What are the primary biological functions of human PLGF2?

Human PLGF2 serves as a key molecule in angiogenesis and vasculogenesis, particularly during embryogenesis. As a member of the VEGF (vascular endothelial growth factor) sub-family, PLGF2 actively stimulates endothelial cell growth, proliferation, and migration. The main source during pregnancy is the placental trophoblast, though it's expressed in many other tissues including villous trophoblast. PLGF2 binds to VEGFR-1 and uniquely to NRP1/neuropilin-1 and NRP2/neuropilin-2 in a heparin-dependent manner. It promotes vascular sprouting, granulation tissue formation during wound healing, and can stimulate tumor cell growth. Research has also demonstrated its role in the recruitment of monocyte-macrophage cells, albeit with lower activity compared to the PLGF1 isoform .

How can I effectively express and purify human PLGF2 in HEK293 cells?

For effective expression and purification of human PLGF2 in HEK293 cells, researchers should follow these methodological steps:

• Expression vector construction: Design a construct containing the human PLGF2 sequence (AA Leu 19 - Arg 170) with a C-terminal polyhistidine tag to facilitate purification.

• Transfection: Electroporate HEK293 cells with the expression vector (50 μg of plasmid DNA at 250 V/cm and 975 μF is an effective protocol).

• Selection: Supplement culture medium with Geneticin (0.8 mg/ml) 48 hours post-transfection to select stable transfectants.

• Clone screening: After approximately 2 weeks, pick G418-resistant clones, amplify them, and screen by ELISA to determine PLGF2 concentration in the medium. Combining the three highest-expressing clones helps avoid clonal effects .

• Purification: Use affinity chromatography targeting the His-tag for protein purification.

• Quality control: Verify protein purity (target ≥95%) using SDS-PAGE and confirm glycosylation status, which causes migration at 28-33 kDa despite the calculated 18.2 kDa molecular weight .

What experimental methods can detect hypoxia-induced PLGF2 expression in endothelial cells?

Detecting hypoxia-induced PLGF2 expression in endothelial cells requires multiple complementary techniques:

• mRNA quantification: Real-time PCR can measure PLGF mRNA upregulation, which shows significant increases after hypoxia exposure. Studies have demonstrated ~9.6-fold increases compared to normoxic conditions after 16 hours of hypoxia (1% O₂) in both HUVEC and H5V cell lines, with maintained expression levels (~8.6- and ~10.5-fold increases) at 24 hours .

• Protein secretion assays: ELISA quantification of PLGF in culture medium reveals significant increases of ~4.6-fold in HUVECs and ~11.5-fold in H5V cells after 24 hours of hypoxia compared to normoxic conditions .

• Western blot analysis: This confirms the effectiveness of hypoxic conditions by detecting increases in HIF-1α and HIF-2α, which are upstream regulators of PLGF expression .

• Chromatin immunoprecipitation (ChIP): This technique is critical for examining the molecular mechanisms of hypoxia-induced PLGF expression, revealing that HIF-1α (but not HIF-2α) directly binds to hypoxia-responsive elements (HREs) in the second intron of the PLGF gene under hypoxic conditions .

How does proteolytic processing affect PLGF2's biological activity in experimental models?

Proteolytic processing significantly alters PLGF2's biological activity in experimental models:

• Plasmin processing generates a protease-resistant core fragment that retains the VEGFR-1 binding site but lacks the carboxyl-terminal domain encoding the heparin-binding domain and an 8-amino acid peptide encoded by exon 7 .

• Functional impact: The angiogenic responses induced by different PLGF forms are distinctly affected by proteolytic processing. While intact PLGF2 effectively increases endothelial cell chemotaxis, vascular sprouting, and granulation tissue formation upon skin injury, these activities are significantly abrogated following plasmin digestion .

• Experimental models: Researchers have generated a truncated PLGF118 isoform mimicking plasmin-processed PLGF to explore its biological function in comparison with PLGF-1 and PLGF-2, enabling the systematic investigation of proteolytic regulation of PLGF activities .

• Measurement approaches: Evaluating these differential activities requires multiple experimental readouts, including endothelial cell migration assays, vascular sprouting assays, and in vivo wound healing models that measure granulation tissue formation .

How do epigenetic mechanisms control hypoxia-induced PLGF2 expression?

Epigenetic mechanisms play a crucial role in controlling hypoxia-induced PLGF2 expression through several interrelated processes:

• Histone acetylation patterns: Under hypoxic conditions (1% O₂), hyperacetylation of histones H3 and H4 occurs specifically in the second intron region of the PLGF gene. This hyperacetylation creates an accessible chromatin structure that facilitates transcription factor binding .

• HIF-1α binding specificity: ChIP analyses reveal that HIF-1α directly binds to the hyperacetylated region containing hypoxia-responsive elements (HREs) in the PLGF second intron. Importantly, HIF-2α does not interact with this PLGF second intron HRE, indicating a selective role for HIF-1α in PLGF regulation .

• Species conservation: This epigenetic regulatory mechanism is conserved between human and mouse PLGF genes, with both showing increased histone acetylation in the second intron region and HIF-1α binding specifically to this region under hypoxia .

• Promoter versus intronic regulation: Despite the presence of putative HREs in the PLGF promoter regions, these sites are not differentially acetylated under hypoxia and do not recruit HIF-1α or HIF-2α, demonstrating that the intronic regulatory elements are specifically involved in hypoxic induction .

How can PLGF2 variants be utilized as dominant negatives in angiogenesis research?

PLGF2 variants offer powerful tools as dominant negatives in angiogenesis research through several mechanisms:

• PLGF2-DE variant design: By substituting amino acid residues D72 and E73 with alanine, researchers have created a PLGF2-DE variant that is unable to bind and activate VEGFR-1 but still retains the ability to heterodimerize with VEGF-A .

• Heterodimerization effects: When overexpressed in VEGF-A producing human tumor cell lines (such as A2780 ovarian carcinoma cells), PLGF2-DE significantly reduces VEGF-A homodimer production through heterodimerization, effectively sequestering VEGF-A .

• Tumor growth inhibition: This PLGF2-DE variant demonstrates strong inhibition of xenograft tumor growth and associated neoangiogenesis, along with significant reduction of monocyte-macrophage infiltration .

• Comparison with wild-type effects: Interestingly, overexpression of wild-type PLGF2 reduces VEGF-A homodimer production similarly to PLGF2-DE through heterodimer formation, but does not inhibit tumor growth and vessel density. This suggests that active PLGF2 homodimers and VEGF-A/PLGF2 heterodimers can compensate for the reduction in VEGF-A homodimers .

• Isoform independence: The 'dominant negative' effect of PLGF-DE variants acts independently of which PLGF isoform is utilized, making this a versatile research tool .

What methodologies can distinguish between PLGF2 homodimer and VEGF-A/PLGF2 heterodimer effects?

Distinguishing between PLGF2 homodimer and VEGF-A/PLGF2 heterodimer effects requires sophisticated methodological approaches:

• Differential expression systems: Researchers can design experimental systems expressing only PLGF2 (producing exclusively homodimers) or co-expressing PLGF2 and VEGF-A (generating both homodimers and heterodimers) in cell lines like A2780 ovarian carcinoma cells .

• Protein quantification: ELISA assays can quantify the reduction in VEGF-A homodimers resulting from heterodimerization with PLGF2, comparing wild-type PLGF2 and variants like PLGF2-DE .

• Functional readouts: Researchers should assess multiple parameters including:

  • Tumor growth rates in xenograft models

  • Vessel density quantification using endothelial markers like CD31

  • Monocyte-macrophage infiltration using markers such as F4/80

  • Endothelial cell chemotaxis

  • Vascular sprouting assays

• Use of variant forms: PLGF2-DE variants that cannot activate VEGFR-1 but can still form heterodimers with VEGF-A provide a powerful tool to specifically study heterodimer formation effects separate from PLGF2 signaling effects .

How do PLGF1 and PLGF2 differ in their effects on monocyte-macrophage recruitment?

PLGF1 and PLGF2 demonstrate significant differences in their effects on monocyte-macrophage recruitment:

• Recruitment efficiency: PLGF2 shows lower activity in recruiting monocyte-macrophage cells compared to the PLGF1 isoform, despite both binding to VEGFR-1, which is involved in monocyte chemotaxis .

• Solubility differences: PLGF2 is characterized as "less soluble" than PLGF1, likely due to its heparin-binding domain that allows interaction with extracellular matrix components. This reduced solubility may explain its limited potential in monocyte-macrophage recruitment compared to the fully soluble PLGF1 .

• Experimental evidence: In comparative studies of tumor models overexpressing either PLGF1 or PLGF2, PLGF1 demonstrates stronger recruitment of bone marrow-derived cells. This functional difference represents a unique distinction between these isoforms beyond their differential binding to neuropilins .

• Quantification approaches: The differential recruitment can be assessed using immunohistochemical staining for markers like F4/80 in tumor sections, providing quantitative measures of macrophage infiltration in response to different PLGF isoforms .

What challenges exist in interpreting conflicting data on PLGF2's role in tumor growth?

Researchers face several challenges when interpreting conflicting data regarding PLGF2's role in tumor growth:

• Variant-dependent effects: Different forms of PLGF2 produce opposite effects on tumor growth. Wild-type PLGF2 may not inhibit tumor growth despite reducing VEGF-A homodimer availability, while the PLGF2-DE variant strongly inhibits xenograft tumor growth and angiogenesis .

• Compensatory mechanisms: The expression of active PLGF2 homodimers and VEGF-A/PLGF2 heterodimers can rescue pro-angiogenic activity lost through reduction of VEGF-A homodimers, complicating interpretation of experimental results .

• Cell-specific responses: Different cell types may respond differently to PLGF2 stimulation due to varying receptor expression profiles, creating apparently conflicting results across different experimental models .

• Proteolytic processing: PLGF2 undergoes proteolytic processing by plasmin, which abrogates its angiogenic activities. Varying levels of proteases in different experimental systems can produce contradictory results regarding PLGF2's effects .

• Immune cell infiltration: PLGF2 affects monocyte-macrophage infiltration, which itself can influence tumor growth through complex immune-mediated mechanisms. This creates another variable that can lead to apparently conflicting tumor growth data .

What are the latest methodological approaches for studying PLGF2 in hypoxic environments?

Studying PLGF2 in hypoxic environments requires state-of-the-art methodological approaches:

• Controlled hypoxic chambers: Modern systems allow precise maintenance of 1% O₂ conditions for cultured human and mouse endothelial cells with tight temporal control, enabling examination of hypoxic responses at specific timepoints (16h, 24h) .

• Multilevel expression analysis: Combining mRNA quantification (qRT-PCR) with protein quantification (ELISA) provides comprehensive understanding of transcriptional and translational regulation of PLGF2 under hypoxia .

• Epigenetic profiling: Chromatin immunoprecipitation (ChIP) analyses using antibodies against histone modifications (H3/H4 acetylation) and hypoxia-inducible factors (HIF-1α, HIF-2α) reveal the epigenetic mechanisms controlling hypoxia-induced PLGF2 expression .

• Gene-region specific analysis: Comprehensive examination of multiple regions across the PLGF gene, including promoter regions and introns, is necessary to identify the specific regulatory elements responsible for hypoxic induction .

• Species-comparative approaches: Parallel analysis of human and mouse systems provides insights into evolutionarily conserved regulatory mechanisms, with evidence indicating that both species exhibit similar patterns of histone acetylation and HIF-1α binding in the second intron under hypoxia .

What are promising therapeutic applications of PLGF2 variants in cancer research?

PLGF2 variants show significant promise for therapeutic applications in cancer research:

• Dominant negative approach: The PLGF2-DE variant (with D72A and E73A substitutions) offers a powerful strategy for inhibiting tumor angiogenesis by sequestering VEGF-A through heterodimerization while preventing compensatory signaling through VEGFR-1 .

• Dual-targeting potential: Engineered PLGF2 variants could simultaneously target multiple angiogenic pathways by modifying both VEGF-A availability and monocyte-macrophage recruitment, potentially overcoming resistance mechanisms seen with single-target approaches .

• Plasmin-resistant variants: Developing PLGF2 variants resistant to plasmin cleavage could provide more stable and predictable therapeutic agents, as proteolytic processing significantly alters PLGF2's biological activities .

• Combination strategies: PLGF2 variants could complement existing anti-angiogenic therapies by targeting distinct aspects of tumor vascularization, potentially enhancing efficacy without increasing toxicity .

• Delivery systems: Development of targeted delivery systems for PLGF2 variants could improve their efficacy and reduce off-target effects, making them more suitable for clinical applications .

How might emerging techniques in protein engineering enhance PLGF2 research applications?

Emerging protein engineering techniques offer significant opportunities to enhance PLGF2 research applications:

• Site-specific glycoengineering: Controlling glycosylation patterns of recombinant PLGF2 could help standardize the 28-33 kDa glycosylated forms currently observed in HEK293 expression systems, reducing variability in experimental results .

• Domain-swapping approaches: Creating chimeric proteins that combine domains from different PLGF isoforms could help identify the precise structural elements responsible for specific biological activities, particularly the differential effects on monocyte-macrophage recruitment .

• Biosensor development: Engineering PLGF2 variants with incorporated fluorescent or bioluminescent reporters could enable real-time monitoring of protein activity, processing, and heterodimerization in living systems .

• Controllable proteolytic sensitivity: Designing PLGF2 variants with engineered sensitivity to specific proteases could create tools for temporal and spatial control of PLGF2 activity in research models .

• Expression system optimization: Further refinement of HEK293 expression systems could improve yield and consistency of recombinant PLGF2 production while maintaining proper post-translational modifications essential for biological activity .