PLGF1 Human, 132 a.a.

What is PLGF1 Human and what are its structural characteristics?

PLGF1 Human (Placental Growth Factor-1) is a growth factor that plays an active role in angiogenesis and endothelial cell growth, stimulating both proliferation and migration of these cells. It is a member of the Vascular Endothelial Growth Factor (VEGF) family and specifically binds to receptor VEGFR-1/FLT1 . The human PLGF1 recombinant protein produced in E. coli is characterized as a non-glycosylated, disulfide-linked homodimer consisting of two 132 amino acid polypeptide chains, with a total molecular mass of approximately 29.7 kDa . The amino acid sequence of PLGF1 is: MLPAVPPQQW ALSAGNGSSE VEVVPFQEVW GRSYCRALER LVDVVSEYPS EVEHMFSPSC VSLLRCTGCC GDENLHCVPV ETANVTMQLL KIRSGDRPSY VELTFSQHVR CECRPLREKM KPERCGDAVP RR, which confers its specific functional properties . The plgf gene is located on chromosome 14q.14 and encodes four isoforms of PLGF, with PLGF-1 and PLGF-2 being the most abundant during pregnancy .

How does PLGF1 differ from other PLGF isoforms?

PLGF exists in four distinct isoforms (PLGF-1, PLGF-2, PLGF-3, and PLGF-4), each with unique structural elements that influence their biological activities. While PLGF-1 shows 53% homology in structure to VEGF-A, there are significant structural differences between the various isoforms . Notably, PLGF-2 and PLGF-4 possess additional heparin-binding domains that are absent in PLGF-1, which affects their interaction with extracellular matrix components and cellular receptors . During pregnancy, PLGF-1 and PLGF-2 are the predominant isoforms and are prominently expressed in villous trophoblast cells . The 132 amino acid structure of PLGF1 forms a homodimer through disulfide linkages, which is essential for its biological activity, whereas other isoforms may have different dimerization properties affecting their function. Understanding these structural differences is crucial for researchers designing experiments targeting specific PLGF isoforms or investigating isoform-specific functions.

What are the optimal storage and handling conditions for recombinant PLGF1?

For optimal preservation of recombinant PLGF1 activity in research applications, proper storage and handling procedures are essential. Lyophilized PLGF1, while stable at room temperature for up to three weeks, should ideally be stored desiccated below -18°C for long-term stability . Upon reconstitution, PLGF1 maintains stability when stored at 4°C for a period of 2-7 days, but for extended storage periods, temperatures below -18°C are recommended . The reconstitution process is critical: it is advised to reconstitute lyophilized Placental Growth Factor-1 in sterile 18MΩ-cm H₂O at a concentration not less than 100μg/ml, which can then be further diluted to prepare working solutions in appropriate aqueous buffers . Researchers should be particularly vigilant about avoiding freeze-thaw cycles, as these can substantially degrade protein quality and compromise experimental outcomes . For reconstitution, the product is typically provided as a sterile filtered white lyophilized powder, prepared from a 0.2μm filtered concentrated solution in 10mM Sodium Phosphate at pH 7.5, which maintains the protein in its most stable configuration .

How can PLGF1 be effectively used in monocyte chemotaxis assays?

Monocyte chemotaxis assays using PLGF1 require precise methodological considerations to generate reliable and reproducible results. The optimal concentration range for PLGF1 in chemotaxis assays has been established between 1.0-10.0 ng/ml, based on its demonstrated ability to effectively chemoattract human monocytes . To perform such assays, researchers typically employ a modified Boyden chamber setup with a porous membrane (5-8 μm pore size) separating the upper compartment containing monocytes from the lower compartment containing PLGF1. The assay should begin with isolation of fresh human monocytes using density gradient centrifugation followed by negative selection to ensure high purity, as contaminating cells can confound results. For optimal experimental design, a concentration gradient should be tested (e.g., 0.1, 1.0, 5.0, and 10.0 ng/ml) alongside appropriate positive controls (such as MCP-1) and negative controls. Incubation times typically range from 2-4 hours at 37°C in a humidified 5% CO₂ atmosphere, with migration quantified either through direct counting of cells that have traversed the membrane or through automated fluorescence-based detection methods for higher throughput. Data interpretation should account for both the chemotactic index (ratio of cells migrating toward PLGF1 versus random migration) and the dose-response relationship across the concentration range.

How do PLGF1 levels correlate with pregnancy complications and what thresholds are clinically significant?

Placental Growth Factor-1 levels demonstrate a complex correlation with pregnancy complications, particularly preeclampsia, with threshold values offering potentially valuable predictive information. Research has established that women who ultimately develop preeclampsia typically exhibit lower PlGF levels early in pregnancy compared to those with uncomplicated pregnancies . In a secondary analysis of the MFMU High-Risk Aspirin study, researchers identified a threshold PlGF value of 87.71 pg/ml (collected between 13w0d–16w6d) for the prediction of preeclampsia, though this demonstrated modest sensitivity and specificity (63% and 66%, respectively) . Interestingly, contrary to expectations, preeclampsia was observed in 21.3% of women with "high" baseline PlGF (mean 163 pg/ml) and in 19.3% of women with "low" PlGF (mean 58.7 pg/ml) . The correlation becomes more complex when factoring in treatments like low-dose aspirin (LDA), where women with low PlGF who received LDA actually showed higher preeclampsia rates (21.74%) compared to those who received placebo (15.91%) . These findings highlight the multifaceted nature of PLGF's role in preeclampsia pathophysiology and suggest that absolute thresholds may be insufficient for clinical prediction without considering other factors such as maternal characteristics, gestational age, and concurrent biomarkers.

Table 1: Relationship Between PLGF Levels and Preeclampsia Risk Based on MFMU Study

What experimental approaches are recommended for studying PLGF1-VEGFR-1 interactions?

Studying the interaction between PLGF1 and VEGFR-1 (FLT1) requires sophisticated experimental approaches that span from molecular binding assays to functional cellular studies. Surface Plasmon Resonance (SPR) provides a powerful method for determining binding kinetics, where purified VEGFR-1 extracellular domain is immobilized on a sensor chip and varying concentrations of PLGF1 are flowed over the surface to measure association and dissociation rates. Complementary to SPR, Isothermal Titration Calorimetry (ITC) can quantify the thermodynamic parameters of the interaction, providing insights into binding enthalpy and entropy that inform on the energetic basis of the interaction. For cellular studies, researchers should employ receptor phosphorylation assays using cells that express VEGFR-1 (such as HUVECs or engineered cell lines) treated with varying concentrations of PLGF1 (1-100 ng/ml), followed by immunoprecipitation and Western blotting for phosphotyrosine residues. Competitive binding assays with VEGF-A are crucial to understand the overlapping binding sites, as PLGF1 and VEGF-A compete for binding to VEGFR-1. Advanced imaging approaches, including FRET (Förster Resonance Energy Transfer) between fluorescently labeled PLGF1 and VEGFR-1, can visualize interactions in real-time within living cells. For functional readouts of this interaction, researchers should measure downstream signaling pathways activated by PLGF1 binding, including ERK/MAPK phosphorylation, PI3K/Akt activation, and calcium flux using appropriate reporter systems or phospho-specific antibodies.

How has the understanding of PLGF1's role in pregnancy evolved through methodological advances?

The understanding of PLGF1's role in pregnancy has evolved significantly through methodological advances, transforming from basic correlative observations to sophisticated predictive algorithms. Early research focused primarily on establishing baseline variations in PlGF levels throughout normal pregnancy, but technological advancements in protein detection and quantification methods have enabled more nuanced investigations into PlGF's predictive capabilities for pregnancy complications . A significant methodological milestone came with the development of algorithms incorporating maternal factors, mean arterial pressure (MAP), uterine artery pulsatility index (UtPI), and PlGF measurements for first-trimester prediction of preeclampsia . These combined screening approaches have demonstrated impressive detection rates, with one algorithm showing a 76.6% detection rate for preeclampsia when incorporating maternal factors, MAP, UtPI, PlGF, and serum pregnancy-associated plasma protein A (PAPP-A) levels . Further refinement through prospective data collection from 35,948 women updated the original algorithm and assessed its performance, revealing detection rates of 75% for preterm preeclampsia and 47% for term preeclampsia, with a 10% false-positive rate . Interestingly, subsequent studies determined that including PAPP-A to models already containing PlGF did not improve screening performance, a finding replicated by multiple researchers, with systematic reviews confirming that PlGF is superior to PAPP-A in first-trimester prediction of preeclampsia . These methodological advances have shifted clinical practice toward implementing PlGF-based testing using commercially available assays to accelerate the diagnosis of preeclampsia in symptomatic women beyond 20 weeks' gestation .

What are key considerations for designing in vitro angiogenesis assays using PLGF1?

Designing robust in vitro angiogenesis assays with PLGF1 requires careful consideration of multiple experimental parameters to ensure physiologically relevant and reproducible results. The selection of endothelial cell type is paramount, with human umbilical vein endothelial cells (HUVECs) commonly used, though tissue-specific endothelial cells (such as dermal or pulmonary microvascular cells) may be more appropriate depending on the research question. Concentration optimization is essential, with researchers typically testing a range from 1-100 ng/ml of PLGF1, using dose-response curves to identify the effective concentration for specific endothelial cell types. The assay format significantly impacts results: tube formation assays on Matrigel provide rapid assessment of network formation (4-24 hours), while spheroid-based sprouting assays better recapitulate the three-dimensional sprouting process over longer periods (24-72 hours). For proliferation studies, endothelial cells should be serum-starved (0.5-1% serum) for 6-12 hours before PLGF1 treatment to minimize the confounding effects of serum growth factors. Positive controls (VEGF-A at 10-50 ng/ml) and negative controls (basal media without growth factors) must be included in all experiments for proper result interpretation. Time-course analyses are crucial to distinguish between early (0-6 hours) and late (24-72 hours) angiogenic responses, as these may involve different signaling mechanisms. Finally, complementary readouts should be employed to comprehensively assess angiogenesis, including quantification of total tube length, number of branch points, and closed network loops for tube formation assays, alongside proliferation (BrdU incorporation, Ki67 staining) and migration assessments (scratch wound or Boyden chamber assays).

What purification techniques are recommended for obtaining high-quality PLGF1 for research?

Obtaining high-quality PLGF1 for research applications requires implementing a strategic combination of expression systems and purification techniques to ensure optimal protein activity and purity. For expression, Escherichia coli remains a popular system for producing recombinant PLGF1, offering advantages in scalability and cost-effectiveness, though researchers must address challenges related to proper disulfide bond formation in this prokaryotic system . Following expression, a multi-step purification protocol is typically employed, beginning with initial capture using immobilized metal affinity chromatography (IMAC) if the construct contains a histidine tag, or ion exchange chromatography exploiting PLGF1's isoelectric properties. Subsequent purification steps should include size exclusion chromatography to separate the correctly formed PLGF1 homodimer (approximately 29.7 kDa) from monomers and higher-order aggregates that may compromise functional studies . Quality assessment is critical at each purification stage, with reversed-phase HPLC and SDS-PAGE analysis under both reducing and non-reducing conditions recommended to verify purity exceeding 95% . For applications requiring exceptionally high purity, affinity chromatography using immobilized VEGFR-1 extracellular domain can further select for functionally active PLGF1 molecules. The final purified product should be formulated in a stabilizing buffer such as 10 mM sodium phosphate (pH 7.5) to maintain structural integrity, followed by sterile filtration through a 0.2 μm filter and lyophilization for long-term storage . Researchers should validate the functional activity of purified PLGF1 using bioassays such as monocyte chemotaxis or endothelial cell proliferation before employing the protein in sophisticated research applications.

What are the recommended methods for quantifying PLGF1 in biological samples?

Accurate quantification of PLGF1 in biological samples requires selecting appropriate analytical methods based on sample type, expected concentration range, and required sensitivity. Enzyme-Linked Immunosorbent Assay (ELISA) remains the gold standard for PLGF1 quantification in serum, plasma, and tissue culture supernatants, with commercial kits offering detection limits as low as 7 pg/ml and dynamic ranges typically spanning 15-1000 pg/ml. For high-throughput applications in clinical settings, automated immunoassay platforms using electrochemiluminescence or chemiluminescence have been validated for PLGF measurements, offering advantages in standardization across laboratories and reduced inter-operator variability. When analyzing PLGF1 in complex tissue samples, Western blotting provides qualitative assessment and molecular weight confirmation, while quantitative analysis requires preliminary extraction steps and comparison to standard curves of recombinant PLGF1. Mass spectrometry-based approaches, particularly multiple reaction monitoring (MRM), offer exceptional specificity for PLGF1 quantification in complex matrices and can simultaneously identify and quantify different PLGF isoforms, though these methods require specialized equipment and expertise. For spatial localization of PLGF1 in tissues, immunohistochemistry or immunofluorescence using validated antibodies against PLGF1 provide important contextual information about expression patterns that complement quantitative measurements. When implementing any quantification method, researchers must establish appropriate sample collection and processing protocols, as factors such as clotting time for serum, anticoagulant choice for plasma, and storage conditions significantly impact measured PLGF1 levels in biological samples.

How is PLGF1 being investigated in conditions beyond preeclampsia?

PLGF1 research has expanded significantly beyond preeclampsia into diverse pathological conditions, reflecting its fundamental role in angiogenesis and vascular homeostasis. In cardiovascular research, PLGF1 is being investigated as both a biomarker and therapeutic target in atherosclerosis, where it influences macrophage accumulation and inflammatory responses within atherosclerotic plaques. Studies have demonstrated that PLGF modulates arterial wall inflammatory responses through monocyte chemotaxis, with experimental administration of recombinant PLGF1 (at concentrations of 1.0-10.0 ng/ml) shown to significantly increase monocyte migration in controlled laboratory settings . In oncology, PLGF1's contribution to tumor angiogenesis has prompted investigations into anti-PLGF strategies as potential cancer therapeutics, with particular interest in tumors that develop resistance to VEGF-targeted therapies. Chronic inflammatory diseases represent another emerging area, with researchers exploring PLGF1's role in conditions such as rheumatoid arthritis, where abnormal angiogenesis contributes to synovial hyperplasia and joint destruction. Ophthalmological applications are gaining prominence, with PLGF1 implicated in pathological angiogenesis in diabetic retinopathy and age-related macular degeneration, prompting clinical trials of anti-PLGF agents for these conditions. Additionally, PLGF1's involvement in tissue repair and regeneration has opened new research directions in wound healing and ischemic tissue recovery, where recombinant PLGF1 shows therapeutic potential for enhancing vascularization in compromised tissues.

What novel methodological approaches are being developed for PLGF1 research?

Novel methodological approaches for PLGF1 research are advancing rapidly across multiple technical domains, offering unprecedented insights into this protein's functions and interactions. Single-cell transcriptomics is revolutionizing our understanding of PLGF1 expression patterns by revealing cell-specific production profiles within heterogeneous tissues such as the placenta, allowing researchers to identify previously unrecognized cellular sources and regulatory mechanisms that are masked in bulk tissue analyses. CRISPR-Cas9 genome editing technologies are enabling precise manipulation of the PLGF gene and its regulatory elements, facilitating the creation of knockout and knock-in models that help delineate isoform-specific functions through controlled expression of individual PLGF variants. Advanced protein engineering approaches are yielding modified PLGF1 variants with enhanced stability, altered receptor binding properties, or incorporation of traceable tags that maintain biological activity while enabling real-time tracking in experimental systems. Microfluidic organ-on-chip platforms represent a significant methodological advancement, allowing researchers to study PLGF1 function in physiologically relevant three-dimensional tissue constructs that better recapitulate the complex multicellular environments of the placenta or vascular networks. Computational biology approaches, including molecular dynamics simulations and systems biology modeling, are providing unprecedented insights into PLGF1-receptor interactions and downstream signaling networks, helping to predict outcomes of experimental manipulations and identify novel therapeutic targets. Additionally, the application of artificial intelligence to analyze large datasets from PLGF1 studies is accelerating pattern recognition and hypothesis generation, particularly in complex clinical scenarios where multiple variables influence PLGF1 levels and effects.