PLGF Human, His

What is the basic structure and molecular characteristics of human PLGF?

Human PLGF is a homodimeric glycoprotein of 46-50 kDa that belongs to the VEGF subfamily. The human PLGF gene is located on chromosome 14q24, spanning an 800-kb-long DNA segment comprising seven exons . The protein exists in four isoforms (PLGF-1, PLGF-2, PLGF-3, and PLGF-4) in humans, with PLGF-1 and PLGF-3 being diffusible isoforms, while PLGF-2 and PLGF-4 contain heparin binding domains . The protein is secreted as a glycosylated homodimer, with different isoforms having distinct properties related to their diffusibility and binding characteristics .

How do the different isoforms of human PLGF differ functionally?

The four human PLGF isoforms have distinct functional characteristics based on their structural differences. PLGF-1 and PLGF-3 are diffusible isoforms that likely act in a paracrine manner, affecting targets at a distance from their production site . In contrast, PLGF-2 and PLGF-4 contain heparin binding domains that cause them to remain cell membrane-associated, suggesting they function in a more localized, autocrine fashion . PLGF-2 has an additional capability of binding to neuropilin (NRP)-1 and -2 receptors due to an insertion of 21 basic amino acids at its carboxyl terminus, providing it with signaling capabilities not present in PLGF-1 . These differences in receptor binding and diffusion properties likely contribute to isoform-specific functions in various physiological and pathological contexts.

What are the primary receptors for human PLGF and how does receptor binding influence signaling?

Human PLGF primarily binds to VEGF receptor-1 (VEGFR-1), also known as fms-related tyrosine kinase-1 (FLT-1), and its soluble variant sFLT-1 . Unlike VEGF, which binds to both VEGFR-1 and VEGFR-2 (KDR/FLK-1), PLGF exhibits specificity for VEGFR-1 . Additionally, PLGF-2 can bind to neuropilin receptor-1 (NP-1) and -2 because of its 21 basic amino acid insertion at the carboxyl terminus . The binding of PLGF to VEGFR-1 initiates signaling pathways that are distinct from those activated by VEGF. Some research suggests that PLGF may enhance pathological angiogenesis by initiating cross-talk between VEGFR-1 and VEGFR-2, though this finding remains controversial . The receptor-binding specificity of PLGF is crucial for understanding its biological functions and developing targeted therapeutic approaches.

What is the normal tissue distribution and expression pattern of PLGF in humans?

PLGF is predominantly expressed in the placenta, which is consistent with its name, but it is also expressed at lower levels in various other tissues . These include the heart, lung, thyroid, liver, skeletal muscle, and bone . During pregnancy, PLGF expression in the placenta corresponds with different stages of placental development, with increased expression in later gestation coinciding with non-branching angiogenesis of the feto-placental circulation and maturation of the utero-placental circulation . PLGF is also expressed in the endometrium during the secretory phase of the menstrual cycle, suggesting a role in embryo implantation . Understanding the tissue-specific expression patterns of PLGF is essential for interpreting its diverse physiological functions and potential roles in pathological conditions.

What are the methodological approaches for studying PLGF expression in different tissues?

Researchers can employ multiple complementary techniques to study PLGF expression in different tissues. For protein-level detection, immunohistochemistry or immunofluorescence using validated anti-PLGF antibodies (such as PL5D11D4) can visualize the spatial distribution of PLGF within tissues . ELISA assays, particularly sandwich ELISAs such as the Human PLGF DuoSet ELISA, can quantify PLGF levels in body fluids or tissue lysates . For mRNA expression analysis, RT-PCR and quantitative real-time PCR are effective for detecting different PLGF isoforms, as demonstrated in studies with extravillous trophoblast cells . Western blotting can confirm protein expression and molecular weight, while recombinant His-tagged PLGF proteins can serve as positive controls . For more complex studies, co-culture systems with human retinal pericytes (HRP) and HREC, or blood vessel/vascular organoids derived from human induced pluripotent stem cells provide three-dimensional models to study PLGF in physiologically relevant contexts .

What role does PLGF play in normal placental development?

PLGF plays a significant role in promoting the development and maturation of the placental vascular system . Studies in PLGF knockout mice have revealed abnormal placental vasculature at implantation sites, characterized by decreased branching in both the anti-mesometrial (feto-placental) vessels and utero-placental vessels, as well as increased lacunarity indicating non-uniform vessel formation . In human placenta, PLGF expression patterns correlate with different stages of placental development, with increased expression in later gestation coinciding with non-branching angiogenesis of the feto-placental circulation and maturation of the utero-placental circulation . Although PLGF knockout mice remain fertile, suggesting redundancy in reproductive mechanisms, the protein clearly influences placental vascular development . Additionally, PLGF may affect extravillous trophoblast (EVT) cell proliferation, as exogenous PLGF-1 has been shown to stimulate EVT cell proliferation in the presence of heparan sulphate proteoglycans, although it does not appear to influence EVT migration or invasiveness .

How is PLGF involved in the pathophysiology of pre-eclampsia?

PLGF has emerged as a critical molecule in the pathophysiology of pre-eclampsia, a pregnancy complication characterized by hypertension and proteinuria. In pre-eclampsia, circulating PLGF levels are significantly decreased while soluble FLT-1 (sFLT-1) levels are elevated . This imbalance creates an anti-angiogenic environment as sFLT-1 acts as a decoy receptor, sequestering PLGF and preventing it from binding to membrane-bound receptors. Multiple animal models have demonstrated that administration of recombinant human PLGF (rhPLGF) can ameliorate pre-eclampsia-like symptoms. In mice transfected with adenovirus to increase sFLT-1, intraperitoneal PLGF-2 administration reduced hypertension . Similarly, in rat reduced uterine placental perfusion (RUPP) models, rhPLGF administered via continuous infusion reduced blood pressure, proteinuria, improved glomerular filtration rate, and decreased markers of oxidative stress . In non-human primate utero-placental ischemic models of pre-eclampsia, rhPLGF reduced both blood pressure and proteinuria . These findings collectively suggest that PLGF replacement or augmentation may represent a potential therapeutic strategy for pre-eclampsia.

What experimental models are available for studying PLGF in disease states?

Researchers have developed several experimental models to study PLGF in disease states, particularly in pre-eclampsia and vascular disorders. Animal models include PLGF knockout mice, which show abnormal placental vasculature and differences in fetal and adult brain development . For pre-eclampsia specifically, the reduced uterine placental perfusion (RUPP) model in rats and the adenovirus-mediated sFLT-1 overexpression model in mice create pre-eclampsia-like states that can be used to test PLGF-related interventions . Non-human primate utero-placental ischemic models provide a closer approximation to human pre-eclampsia . In vitro, human cell co-cultures of endothelial cells and pericytes, such as human retinal pericytes (HRP) and human retinal endothelial cells (HREC), allow for the study of PLGF's effects on vascular interactions . More advanced three-dimensional vascular organoids derived from human induced pluripotent stem cells (iPSCs) offer a sophisticated model system for studying vascular development and pathologies . These diverse models enable researchers to investigate PLGF's role in multiple pathological contexts and test potential therapeutic approaches.

How can PLGF measurements be used for prediction and diagnosis of pre-eclampsia?

PLGF has emerged as an increasingly important biomarker for the prediction, diagnosis, and management of pre-eclampsia . Decreased maternal serum PLGF levels precede the clinical onset of pre-eclampsia by several weeks, making it a valuable early predictor. Quantitative measurement of circulating PLGF using validated ELISA assays, such as the Human PLGF Quantikine ELISA Kit, can provide reliable assessment of PLGF levels in maternal serum or plasma . The diagnostic value of PLGF is enhanced when measured in conjunction with other biomarkers, particularly the sFLT-1/PLGF ratio, which exhibits high sensitivity and specificity for predicting pre-eclampsia. A systematic approach to PLGF testing should consider gestational age-specific reference ranges, as normal PLGF levels vary throughout pregnancy, typically rising until 30 weeks and then declining gradually. For research applications, it's important to standardize sample collection, processing methods, and utilize validated assays with appropriate controls to ensure reproducible results across studies.

What are the considerations when working with His-tagged human PLGF proteins?

When working with His-tagged human PLGF proteins, several methodological considerations are crucial for successful experiments. For purification, immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co2+ resins is most common, but buffer composition must be optimized to minimize non-specific binding while maintaining PLGF stability. The position of the His-tag (N- or C-terminal) may affect protein folding and receptor binding, so functional validation is essential after purification. SDS-PAGE analysis under reducing and non-reducing conditions can verify the dimeric nature of PLGF and the integrity of the His-tag, as demonstrated with Human PLGF protein, His tag (GTX138450-pro) . For immunodetection, researchers should use antibodies specifically validated for His-tagged PLGF to avoid epitope masking by the tag. If the His-tag needs to be removed for certain applications, incorporate protease cleavage sites and optimize cleavage conditions to maintain PLGF activity. Storage conditions are critical—purified His-tagged PLGF typically requires -80°C storage with glycerol as a cryoprotectant, with minimal freeze-thaw cycles to preserve activity.

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

For quantifying PLGF in biological samples, sandwich ELISA is the gold standard method. Commercial kits like the Human PLGF DuoSet ELISA (DY264) provide optimized capture and detection antibody pairings with recommended concentrations . When analyzing complex matrices such as serum or plasma, researchers should evaluate appropriate diluents and potentially develop matrix-specific standard curves to account for matrix effects . For research requiring high sensitivity, chemiluminescent or fluorescent detection systems can provide lower detection limits compared to colorimetric methods. Western blotting can complement ELISA data by confirming the molecular weight and integrity of PLGF. When developing custom quantification assays, researchers should validate antibody specificity for the PLGF isoform of interest, as antibodies may have differential reactivity against PLGF-1, PLGF-2, PLGF-3, and PLGF-4. For complex tissue samples, immunohistochemistry or immunofluorescence with validated anti-PLGF antibodies provides spatial information about PLGF expression and localization that complements quantitative data from ELISAs.

How can cell culture models be optimized for studying PLGF signaling?

Optimizing cell culture models for studying PLGF signaling requires careful consideration of experimental design. When selecting cell lines, researchers should verify endogenous expression of PLGF receptors (VEGFR-1/FLT-1, neuropilins) through Western blotting or flow cytometry, as receptor expression levels can significantly impact experimental outcomes. Co-culture systems, such as endothelial cells with pericytes, provide more physiologically relevant contexts for studying PLGF's effects on vascular interactions . Three-dimensional culture systems, including vascular organoids derived from human induced pluripotent stem cells, offer advanced models that better recapitulate in vivo vascular structures . The presence or absence of heparan sulfate proteoglycans (HSPGs) in the culture environment is critical, as they can sequester PLGF-1 away from its receptor or modify PLGF-receptor interactions . When studying PLGF signaling kinetics, time-course experiments with phospho-specific antibodies against downstream signaling molecules can map activation patterns. For receptor specificity studies, selective receptor blockade using antibodies like anti-VEGFR1 (MF1) can help delineate PLGF-specific signaling pathways .

How does PLGF interact with other members of the VEGF family in complex angiogenic processes?

PLGF exhibits complex interactions with other VEGF family members in angiogenic processes through multiple mechanisms. Unlike VEGF, which binds both VEGFR-1 and VEGFR-2, PLGF binds exclusively to VEGFR-1 and its soluble form sFLT-1 . This receptor specificity creates both competitive and complementary interactions with VEGF. By binding to VEGFR-1, PLGF can displace VEGF, making more VEGF available to activate VEGFR-2, which is the primary mediator of angiogenic effects—a mechanism called the "VEGF displacement hypothesis" . Additionally, PLGF can form heterodimers with VEGF, creating molecules with unique signaling properties distinct from either homodimer. The signaling outcomes of PLGF/VEGFR-1 binding differ from VEGF/VEGFR-1 binding, suggesting ligand-specific activation of downstream pathways. In pathological contexts like pre-eclampsia, the balance between PLGF and soluble FLT-1 (sFLT-1) is particularly important, as sFLT-1 acts as a decoy receptor that can sequester both PLGF and VEGF . The contextual nature of PLGF activity is further demonstrated by studies showing that PLGF knockout mice display impaired angiogenesis in pathological conditions but have normal development, suggesting that PLGF's interactions with other VEGF family members may be particularly important during stress or disease states .

What are the methodological challenges in developing PLGF-targeted therapeutics?

Developing PLGF-targeted therapeutics presents several methodological challenges. The contextual nature of PLGF's activity creates complexity—it appears redundant in normal development (PLGF knockout mice are viable) but important in pathological conditions . This context-dependent activity requires sophisticated models that accurately recapitulate disease environments when screening potential therapeutics. There's contradictory evidence regarding PLGF's role in pathological angiogenesis, with some studies suggesting it enhances pathological angiogenesis through VEGFR-1/VEGFR-2 cross-talk, while others don't confirm these findings . For pre-eclampsia therapeutics, delivery obstacles exist as any treatment must be safe for both mother and fetus while effectively targeting the placental interface. The timing of PLGF-based interventions is critical, as evidenced by animal models where recombinant PLGF administration reduced hypertension and proteinuria . Developing specific antibodies or small molecules that can modulate PLGF activity without affecting other VEGF family members requires advanced screening methodologies and structural biology approaches. For recombinant PLGF therapies, optimizing production of correctly folded, glycosylated protein with appropriate isoform selection presents pharmaceutical development challenges. Finally, establishing reliable biomarkers for patient stratification and therapeutic monitoring is essential for clinical translation of PLGF-targeted approaches.

How might advanced imaging techniques enhance our understanding of PLGF biology in vivo?

Advanced imaging techniques offer transformative potential for understanding PLGF biology in living systems. Real-time in vivo imaging of PLGF activity can be achieved using genetically encoded FRET-based biosensors that report on VEGFR-1 activation, allowing dynamic visualization of PLGF signaling in transgenic animal models. Two-photon intravital microscopy enables deep tissue imaging of labeled PLGF and its interactions with blood vessels in mouse models, providing insights into how PLGF influences vascular remodeling at cellular resolution. For human studies, contrast-enhanced ultrasound with PLGF-targeted microbubbles can assess VEGFR-1 expression levels in tissues of interest, particularly useful for evaluating placental function in pregnancy complications. PET imaging with radiolabeled anti-PLGF antibodies or PLGF peptides allows whole-body assessment of PLGF receptor distribution and occupancy, valuable for both basic research and therapeutic development. Optical coherence tomography angiography (OCTA) provides non-invasive visualization of vascular networks that can be combined with PLGF interventions to assess functional outcomes in vascular remodeling. Light-sheet microscopy of cleared tissues offers comprehensive 3D visualization of PLGF expression patterns and vascular architecture in whole organs or embryos at unprecedented resolution. These complementary imaging approaches, combined with PLGF manipulation through genetic or pharmacological means, can reveal the spatial and temporal dynamics of PLGF activity across different physiological and pathological contexts.