PLGF Human, HEK

What is PlGF and what is its primary biological function?

PlGF is an angiogenic growth factor that functions both in vitro and in vivo. Under normal physiological conditions, human PlGF expression is largely restricted to the placenta . Within the human maternal-fetal interface, PlGF is prominently expressed in villous cytotrophoblast, syncytiotrophoblast, and uterine Natural Killer (uNK) cells .

PlGF promotes angiogenesis (blood vessel formation) and is essential for normal placental development. Evidence from animal models highlights its functional importance during pregnancy. Studies have shown that pups lacking PlGF expression, while viable, have significantly reduced placental and fetal birth weights . Additionally, chronic maternal hypoxia decreases systemic levels of PlGF, increases sflt-1 (soluble PlGF receptor) levels, and results in significant intrauterine growth restriction .

How is PlGF expression regulated under different oxygen conditions?

PlGF expression shows cell-type specific regulation under varying oxygen tensions:

• In trophoblast cells: Hypoxia (low oxygen tension) downregulates PlGF gene expression. Real-time RT-PCR confirms that hypoxia significantly decreases PlGF mRNA expression in JEG-3 cells and primary trophoblast .

• In non-trophoblast cells: Hypoxia increases PlGF mRNA in cells like HeLa . The -1521/+34 promoter clone demonstrated a significant 3.93 ± 1.0 fold increase in transcriptional activity after 24 hours of hypoxia in HeLa cells .

This differential regulation appears to occur at the transcriptional level. Temporally, expression of PlGF mRNA is variably decreased by 3 hours of hypoxia and continues to decrease to 30% of control levels by 6 hours and to 10% by 24 hours of hypoxia in trophoblast cells .

What is the relationship between PlGF and preeclampsia?

PlGF has emerged as an important biomarker for preeclampsia. Previous studies have shown that maternal serum PlGF levels are significantly reduced in preeclampsia and that this decrease occurs before clinical onset of the symptoms . Additionally, the presence of an alternatively spliced, soluble form of the PlGF receptor (sflt-1 or sVEGFR1) is increased in preeclamptic women .

Clinical studies have established important PlGF thresholds for pregnancy assessment:

These data show that low PlGF concentrations (<100 pg/ml) identify women with more marked hypertension, increased adverse maternal outcomes, higher preterm delivery rates, and greater incidence of small-for-gestational-age infants .

What is the role of hypoxia-inducible factor (HIF-1) in PlGF regulation?

Despite initial expectations, research suggests that PlGF transcription is not directly mediated by functional HIF-1 activity in either trophoblast or non-trophoblast cells . This finding is surprising given that:

• Hypoxia induces a 4.4 ± 0.013 fold increase in HIF-1α protein accumulation in the nuclei of hypoxic primary trophoblast and similar increases in JEG-3, hEK-293, and HeLa cell lines .

• The 1.5 Kb region (-1521/+34) and distal subclone (-1521/-650) of the human PlGF promoter both contain two consensus hypoxia response elements (HREs) at positions -1274 and -952 .

• Overexpression of constitutively active or dominant negative HIF-1α in JEG-3 and HeLa cells did not significantly alter transcription patterns of the PlGF gene .

This suggests that different regulatory mechanisms control PlGF expression in hypoxic conditions, independent of the classical HIF-1 pathway.

What regulatory elements control PlGF transcription under hypoxia?

The PlGF gene contains several regulatory elements that may influence its transcription:

How do co-factors and protein interactions regulate PlGF expression?

Research on protein interactions reveals potential regulatory mechanisms for PlGF expression:

• p300/CBP recruitment: HRE oligo-binding assays demonstrated similar patterns of nuclear HIF-1α binding to a consensus HRE oligomer and p300 recruitment to the HRE/HIF-1α complex in hypoxic JEG-3, hEK-293, and HeLa cells . This suggests that differences in basic HIF-1 complex formation are not responsible for the differential PlGF regulation.

• CITED-2 competition: The increase in CITED-2 expression under hypoxia could competitively limit p300/CBP recruitment to PlGF HRE-HIF-1 complexes in trophoblast, potentially contributing to decreased transcription .

• Primary trophoblast vs. cell lines: In primary trophoblast, researchers could not detect HIF-1α binding to the consensus HRE after 24 hours in hypoxia, despite clear downregulation of PlGF mRNA . This finding suggests potential differences between primary cells and cell lines that may be significant for understanding PlGF regulation.

What reporter systems are effective for studying PlGF gene transcription?

For studying PlGF gene transcription, researchers have successfully employed several reporter systems:

• PlGF promoter-reporter constructs: A 1.5 Kb region (-1521/+34) and 2 subregions (-1521/-650, -698/+34) of the human PlGF gene cloned into a β-galactosidase (β-gal) reporter vector pBlue-TOPO® have been used to investigate promoter activity . This approach allows for determination of which regions are essential for transcriptional regulation.

• HRE reporter system: The pGL2-TK-HRE plasmid containing a minimal TK promoter fragment linked to a triple repeat consensus HRE (5'-GTGACTACGTGCTGCCTAG-3') and a firefly luciferase reporter cassette can be used to assess the general hypoxia responsiveness of cell lines . This system showed significant increases under hypoxic conditions in all tested cell lines (JEG-3 = 14.2 ± 4.1 fold, hEK-293 = 19.7 ± 4.6 fold, HeLa = 17.2 ± 6.7 fold) .

• Co-transfection controls: For normalization purposes, co-transfection with control vectors like pSV40-β-gal or RSV-Luc is recommended to account for transfection efficiency differences .

How can researchers assess HIF-1 binding to the PlGF promoter?

To assess HIF-1 binding to hypoxia response elements in the PlGF promoter, researchers can use:

• Biotinylated oligonucleotide binding assay: This method involves using equimolar concentrations of a biotinylated 54-mer oligonucleotide corresponding to a triple repeat of the functional HRE sequence (5' GCCCTACGTGCTGTCTCA 3') . The minimally functional HRE sequence 5'TACGTG 3' within this oligonucleotide is repeated twice within the 1.5 Kb region of the PlGF promoter .

• Avidin-D matrix pull-down: Biotinylated double-stranded HRE is bound to Avidin-D matrix and combined with nuclear extract from either normoxic or hypoxic cells . After washing, Avidin-D beads are re-suspended in 2X Laemmli sample buffer, and proteins in the supernatants are immunoblotted for HIF-1α and p300 .

• Controls: No HIF-1α nor p300 proteins should be co-precipitated by the HRE oligo from nuclear lysates of cells maintained in normoxic conditions (21% O₂), serving as negative controls .

What cell models are most appropriate for studying PlGF regulation?

Based on research findings, several cell models are recommended for studying PlGF regulation:

• JEG-3 cells: This human choriocarcinoma cell line serves as a good model for trophoblast, showing decreased PlGF expression under hypoxia similar to primary trophoblast . They are easier to culture and manipulate than primary cells.

• Primary trophoblast: These cells provide the most physiologically relevant model for studying PlGF in the placenta but may show subtle differences from cell lines, such as less HIF-1α detection in nuclear extracts compared to JEG-3 cells .

• HEK-293 cells: These human embryonic kidney cells represent non-trophoblast cells that show different patterns of PlGF regulation compared to trophoblast cells . They are useful for comparative studies to understand cell-specific regulation.

• HeLa cells: As another non-trophoblast cell line, HeLa cells show increased PlGF expression under hypoxia, providing a clear contrast to trophoblast models .

Using multiple cell types allows researchers to investigate cell-specific regulatory mechanisms and confirm whether observed effects are trophoblast-specific or represent more general cellular responses.

How does PlGF testing improve clinical outcomes in suspected preeclampsia?

PlGF testing has demonstrated several clinical benefits in the management of suspected preeclampsia:

• Faster diagnosis: Time to diagnosis of preeclampsia was significantly lower in the revealed PlGF testing group (1.9 days) compared to usual care (4.1 days) across all PlGF categories (time ratio 0.36, 95% CI 0.15–0.87; p = 0.027) .

• Reduced adverse outcomes: There was a reduction in adverse maternal outcomes in women whose PlGF results were revealed when levels were 12–100 pg/ml compared to usual care (3.8% vs 6.9%; adjusted OR 0.15, 95% CI 0.03–0.92) .

• Improved risk stratification: PlGF < 100 pg/ml identifies women with more marked hypertension, increased adverse maternal outcomes, higher preterm delivery rates, and greater incidence of small-for-gestational-age infants .

The benefit appears particularly pronounced for women with PlGF levels of 12-100 pg/ml, as these may represent cases with silent multi-organ disease who might otherwise go undetected .

What is the diagnostic accuracy of different PlGF thresholds for predicting preeclampsia?

PlGF has high diagnostic accuracy for predicting preeclampsia requiring delivery:

• Low PlGF (<100 pg/ml): Has a high diagnostic accuracy (0.96; 95% confidence interval, 0.89–0.99) and negative predictive value (0.98; 0.93–0.995) for determining preeclampsia requiring delivery within 14 days .

• Very low PlGF (<12 pg/ml): Associated with the highest risk. 78.3-86.2% of women in this category received a final diagnosis of preeclampsia, with 59% delivering within 14 days of testing .

• Normal PlGF (>100 pg/ml): Excellent for ruling out imminent preeclampsia. Only 8% of women with PlGF > 100 pg/ml delivered within 14 days of enrollment .

These findings suggest that PlGF testing can effectively identify women who need increased surveillance and those who can be safely monitored with routine care.

How should researchers interpret PlGF values in different clinical contexts?

When interpreting PlGF values in research settings, several considerations are important:

• Gestational age: PlGF levels naturally vary throughout pregnancy, so results must be interpreted in the context of gestational age.

• Clinical symptoms: The predictive value of PlGF is enhanced when combined with clinical symptoms. For example, among women with suspected preeclampsia (based on symptoms), those with PlGF < 12 pg/ml had mean systolic blood pressure of 150 mmHg versus 136 mmHg in those with PlGF > 100 pg/ml .

• Comorbidities: Other conditions may influence PlGF levels and should be considered when interpreting results.

• Additional biomarkers: Research increasingly focuses on combining PlGF with other biomarkers like sFlt-1 (creating the sFlt-1/PlGF ratio) to improve diagnostic accuracy.

• Time to delivery: Consider that 59% of women with PlGF < 12 pg/ml delivered within 14 days compared to only 8% with PlGF > 100 pg/ml . This time interval is critical for clinical decision-making.

What are the current contradictions in understanding PlGF regulation under hypoxia?

Several contradictions exist in our understanding of PlGF regulation under hypoxia:

• HIF-1 independence: Despite the presence of putative HREs in the PlGF promoter, regulation of PlGF transcription under hypoxic conditions appears independent of HIF-1 in both trophoblast and non-trophoblast cells . This contradicts the canonical model of hypoxia-responsive gene regulation.

• Cell-type specific responses: PlGF shows opposing transcriptional responses to hypoxia in trophoblast versus non-trophoblast cells . This suggests complex regulatory mechanisms that are not fully understood.

• Transcription factor binding: In primary trophoblast, researchers could not detect HIF-1α binding to consensus HRE after 24 hours in hypoxia despite clear downregulation of PlGF mRNA . This suggests either transient binding or alternative regulatory mechanisms.

• Temporal dynamics: The inverse temporal relationship between CITED-2 expression (which increases under hypoxia) and PlGF expression (which decreases) suggests a potential regulatory relationship, but the exact mechanism remains unclear .

These contradictions highlight the need for further research to fully understand the complex regulatory networks controlling PlGF expression.

How might PlGF regulation contribute to the pathophysiology of preeclampsia?

The unique regulation of PlGF likely contributes to preeclampsia pathophysiology through several mechanisms:

• Hypoxia-induced downregulation: Preeclampsia is thought to be associated with reduced placental perfusion and relative hypoxia . The trophoblast-specific decrease in PlGF under hypoxia could contribute to the reduced maternal serum PlGF levels observed in preeclampsia .

• Balance with sFlt-1: Evidence suggests that decreased production of trophoblast PlGF coupled with decreased bioavailability of PlGF (due to increased sFlt-1) likely contribute to the placental, vascular, and renal pathologies commonly associated with preeclampsia .

• Temporal relationship to clinical symptoms: The decrease in maternal serum PlGF occurs before clinical onset of preeclampsia symptoms , suggesting it may be part of the early pathophysiological process rather than simply a consequence.

• Placental insufficiency: Lower PlGF levels correlate with higher rates of small-for-gestational-age infants , indicating a relationship between PlGF dysregulation and placental insufficiency.

What are promising future directions for PlGF research in HEK and trophoblast models?

Several promising research directions could advance our understanding of PlGF biology:

• Single-cell transcriptomics: Applying single-cell RNA sequencing to investigate heterogeneity in PlGF expression and regulation within trophoblast populations and comparing with HEK cells could provide insights into cell-specific regulatory mechanisms.

• Epigenetic regulation: Investigating differences in epigenetic modifications of the PlGF promoter between trophoblast and non-trophoblast cells (like HEK) under different oxygen conditions could reveal mechanisms for cell-specific responses.

• Non-HIF-1 transcription factors: Identifying alternative transcription factors that regulate PlGF expression independently of HIF-1 would help resolve current contradictions in understanding hypoxic regulation.

• CITED-2 mechanisms: Further investigation into how CITED-2 influences PlGF expression could clarify the inverse relationship observed under hypoxia.

• Therapeutic targeting: Developing approaches to modulate PlGF expression or bioavailability in a cell-specific manner could lead to novel interventions for preeclampsia and other pregnancy complications.

• Improved in vitro models: Developing three-dimensional co-culture systems of trophoblast and endothelial cells could better recapitulate the placental environment and provide more physiologically relevant insights into PlGF regulation.