FLT1 Human

What is FLT1 and what is its primary role in human cardiovascular biology?

FLT1, also known as VEGFR1, is a tyrosine kinase receptor predominantly expressed in vascular endothelial cells. It functions as a key regulator of blood vessel development (angiogenesis), cell proliferation, migration, differentiation, and cell survival . In humans, FLT1 is most abundantly expressed in the placenta, with placental FLT1 mRNA levels 40-50 times higher than in other tissues . FLT1 binds vascular endothelial growth factor (VEGF) with high affinity and serves as a critical regulator of vascular development.

The protein exists in both membrane-bound and soluble forms (sFLT1), with the latter functioning as an inhibitor of VEGF signaling. Dysregulation of FLT1 expression or function has been implicated in several pathological conditions, including preeclampsia and coronary artery disease .

How is the FLT1 gene organized and what are its major isoforms?

The human FLT1 gene contains regulatory elements that control its expression in different tissues. Research has identified a highly conserved enhancer within intron 10 of the FLT1 locus termed enFLT1, which directs expression throughout the cardiovascular system . The gene produces multiple isoforms through alternative splicing and polyadenylation.

For FLT1 mRNA, just three isoforms account for more than 94% of all transcripts in the human placenta . These include the full-length membrane-bound form and soluble variants. One soluble isoform, designated sFlt1-14 or sFlt1-e15a, encodes a 733 amino acid (95-135 kDa) protein that is produced primarily by non-endothelial cells, particularly degenerative syncytiotrophoblasts in the placenta . This form appears to be unique to primates and is overproduced during preeclampsia .

What mechanisms regulate FLT1 expression in human tissues?

FLT1 expression is controlled through multiple mechanisms, including:

• Transcriptional regulation: The FLT1 gene contains several regulatory elements that bind transcription factors involved in cardiovascular development. The enhancer element enFLT1 has been shown to be bound by multiple transcription factors, including NKX2-5, which is critical for cardiac development .

• Enhancer elements: The human enFLT1 enhancer directs gene expression throughout the developing cardiovascular system. In transgenic medaka fish models, this enhancer drives GFP reporter expression specifically in cardiovascular tissues .

• Alternative polyadenylation: Polyadenylation site sequencing (PAS-Seq) has revealed that FLT1 produces alternatively polyadenylated mRNA isoforms, particularly in the placenta. These different polyadenylation patterns contribute to the production of soluble FLT1 variants .

• Single nucleotide polymorphisms: Functional SNPs within the FLT1 gene can affect its transcriptional regulation. For example, rs74412485, located in intron 1 of FLT1, enhances its transcriptional level and has been associated with coronary artery disease .

What is the significance of soluble FLT1 (sFLT1) in human pathophysiology?

Soluble FLT1 (sFLT1) plays critical roles in both normal physiology and pathological conditions:

• Normal function: sFLT1 serves as a natural antagonist to VEGF signaling by binding VEGF with high affinity but not transducing signals, thus regulating angiogenesis during development.

• Preeclampsia: sFLT1 is significantly overproduced in preeclampsia, particularly early-onset preeclampsia (symptom onset <34 weeks). These anti-angiogenic factors originate primarily from the placenta and contribute to maternal symptoms . Placental FLT1 mRNA levels strongly correlate with maternal blood pressure, consistent with sFLT1 overproduction being a significant driver of clinical symptoms .

• Biomarker potential: Detection of sFLT1 variants such as Flt1-14 in human serum can serve as a biomarker for preeclampsia. Western blot analysis using specific antibodies can detect these variants in serum samples from preeclamptic patients .

• Therapeutic target: The three predominant FLT1 mRNA isoforms represent potential targets for therapeutic RNA interference (RNAi) in both early and late-onset preeclampsia .

What are the recommended protocols for RNA interference targeting FLT1 in human cells?

Based on published research, the following methodology has proven effective for FLT1 knockdown in human endothelial cells:

• Cell isolation and culture: Human fetoplacental endothelial cells should be isolated from placentas of full-term, uncomplicated pregnancies after scheduled Cesarean section .

• siRNA transfection:

  • Use validated siRNA sequences targeting FLT1

  • Include appropriate control siRNAs (non-targeting)

  • Transfect cells using a lipid-based transfection reagent according to manufacturer's protocols

  • Optimize transfection conditions to minimize toxicity while maximizing knockdown efficiency

• Validation of knockdown efficiency:

  • Confirm FLT1 knockdown at both mRNA level (real-time PCR) and protein level (western blotting)

  • Check for specificity by confirming that other related receptors (e.g., KDR/VEGFR2) are not affected

  • Include full western blot images to demonstrate specificity

• Functional assays: Following confirmed knockdown, cells can be subjected to various functional assays:

  • MTT assay for cell viability/proliferation

  • Wound scratch assay for migration

  • Tube formation assay for angiogenic capacity

  • Western blot analysis of downstream signaling after VEGFA treatment (60 ng/ml)

• Statistical analysis: Perform experiments in triplicate using cells from at least three separate subjects. Use one-way ANOVA with Tukey post-hoc testing for statistical analysis .

How can CRISPR/Cas9 be optimized for studying FLT1 enhancer elements?

CRISPR/Cas9-mediated gene editing has been successfully employed to delete the FLT1 enhancer (enFLT1) to study its function. The following methodology is recommended:

• Guide RNA design:

  • Design guide RNAs flanking the enhancer sequence

  • Ensure minimal off-target effects by using validated prediction tools

  • For the enFLT1 enhancer, design guides targeting the conserved regions identified by bioinformatic analysis

• Cell model selection:

  • Human embryonic stem cells (hESCs) provide a useful model as they can be differentiated into endothelial cells

  • Consider using reporter lines (e.g., NKX2-5(eGFP/wt)) to facilitate monitoring of cardiovascular differentiation

• Validation of enhancer deletion:

  • Confirm deletion by PCR and sequencing

  • Generate homozygous deletion lines

  • Create appropriate control lines (e.g., deletion of exon 1 to serve as a gene knockout control)

• Functional characterization:

  • Differentiate edited stem cells into endothelial cells

  • Perform RNA sequencing to identify differentially expressed genes compared to wildtype cells

  • Conduct gene ontology analysis to identify affected pathways

  • Compare enhancer deletion effects with those of gene deletion

• In vivo validation:

  • Consider using model organisms like medaka fish for in vivo validation

  • Create transgenic lines expressing reporter genes under the control of the human enhancer

  • Evaluate phenotypic effects of enhancer deletion on vascular development

What are the most reliable methodologies for measuring sFLT1 levels in clinical samples?

Several methodologies can be employed to reliably measure sFLT1 levels in clinical samples:

• Western blotting:

  • Use specific antibodies targeting FLT1 variants (e.g., Human VEGFR1/Flt-1 Variant Flt1-14 Antibody)

  • Optimize for non-reducing conditions for certain variants

  • Prepare samples carefully to avoid protein degradation

  • Include appropriate controls (e.g., preeclamptic serum as positive control)

• ELISA:

  • Commercial ELISA kits specifically designed for sFLT1 detection provide quantitative measurement

  • Ensure the kit can detect the specific isoforms of interest

  • Follow manufacturer's protocols carefully, particularly regarding sample preparation

• Mass spectrometry:

  • For more detailed analysis of specific sFLT1 variants

  • Requires specialized equipment but provides high specificity and sensitivity

  • Useful for distinguishing between closely related isoforms

• Polyadenylation site sequencing (PAS-Seq):

  • For research purposes to identify and quantify alternatively polyadenylated FLT1 mRNA isoforms

  • Provides insights into the relative abundance of different isoforms in tissues

  • Has been successfully applied to normal and preeclamptic placental tissues

• Real-time PCR:

  • Design primers specific to FLT1 variants

  • Use for relative quantification of FLT1 isoform expression

  • Particularly useful for tissue samples rather than serum

How do genetic variations in the FLT1 gene impact vascular disease risk?

Genetic variations in FLT1 have been associated with vascular disease risk, particularly coronary artery disease (CAD):

• Single nucleotide polymorphisms (SNPs):

  • rs9319428 in FLT1 has shown a convincing association with CAD in a Japanese population (P=5.98 × 10^-8)

  • Further fine mapping revealed that another SNP (rs74412485) had an even more profound genetic effect for CAD (P=2.85 × 10^-12)

  • This SNP is located in intron 1 of FLT1 and enhances the transcriptional level of the gene

• Functional consequences of genetic variations:

  • The functional SNP rs74412485 leads to increased expression of FLT1

  • This genetically coded increase in FLT1 expression may activate inflammatory cascades that contribute to CAD development

  • RNA interference experiments have shown that suppression of FLT1 results in decreased expression of inflammatory adhesion molecules

• Tissue-specific effects:

  • FLT1 expression has been observed in endothelial cells of human coronary arteries

  • The impact of genetic variations may be more pronounced in specific vascular beds

  • The heritability factor for CAD is estimated at 40-50%, suggesting significant genetic contributions

What are the current challenges in investigating FLT1's role in preeclampsia?

Researchers face several challenges when investigating FLT1's role in preeclampsia:

• Complexity of FLT1 regulation:

  • Multiple isoforms with distinct functions

  • Complex regulatory networks involving enhancers and other cis-regulatory elements

  • Tissue-specific expression patterns that may vary during pregnancy

• Heterogeneity of preeclampsia:

  • Early-onset PE (EO-PE; symptom onset <34 weeks) and late-onset PE (LO-PE; symptom onset >34 weeks) show distinct gene expression profiles

  • Different pathophysiological mechanisms may be involved in different preeclampsia subtypes

• Methodological challenges:

  • Need for appropriate cellular models that recapitulate placental function

  • Difficulty in obtaining human placental samples at early stages of disease

  • Challenges in creating animal models that fully mimic human preeclampsia

• Therapeutic targeting:

  • Developing specific inhibitors for sFLT1 without affecting membrane-bound FLT1 function

  • Designing RNAi approaches that target specific isoforms

  • Determining the optimal timing for intervention

• Biomarker validation:

  • Establishing reliable cutoff values for sFLT1 levels in different populations

  • Integrating sFLT1 measurements with other biomarkers for improved prediction

  • Standardizing measurement techniques across clinical laboratories

How do FLT1-mediated signaling pathways differ between normal and pathological conditions?

FLT1 signaling shows distinct patterns between normal and pathological states:

• Normal signaling:

  • FLT1 functions as a regulator of blood vessel development and angiogenesis

  • It maintains balance between pro-angiogenic and anti-angiogenic factors

  • Soluble FLT1 serves as a natural VEGF antagonist to fine-tune angiogenic responses

  • FLT1 regulates extracellular matrix organization and blood vessel morphogenesis

• Pathological signaling in preeclampsia:

  • Excessive production of sFLT1 disrupts angiogenic balance

  • Three predominant FLT1 mRNA isoforms account for >94% of all transcripts and are upregulated

  • Increased transcription of the entire FLT1 locus occurs in both early-onset and late-onset preeclampsia

  • sFLT1 acts as a "VEGF sponge," preventing normal VEGF signaling

• Pathological signaling in coronary artery disease:

  • Increased FLT1 expression due to functional SNPs activates inflammatory cascades

  • RNA interference experiments show that suppression of FLT1 decreases expression of inflammatory adhesion molecules

  • The inflammatory response contributes to atherosclerotic processes and CAD development

• Regulatory networks:

  • Deletion of the FLT1 enhancer (enFLT1) affects 211 differentially expressed genes

  • Most of these genes (179) overlap with those affected by FLT1 gene deletion

  • Affected pathways include organelle fission, extracellular structure organization, and ECM composition regulation

What are the best approaches for studying FLT1 enhancer activity in different tissue contexts?

To effectively study FLT1 enhancer activity across different tissue contexts, researchers should consider these methodological approaches:

• Reporter gene assays in transgenic models:

  • Clone the human enhancer sequence (enFLT1) into reporter vectors (e.g., modified ZED vector)

  • Generate stable transgenic lines in model organisms like medaka fish

  • Characterize reporter gene expression patterns throughout development

  • This approach has successfully demonstrated that human enFLT1 drives GFP expression throughout the cardiovascular system in medaka

• In vitro enhancer activity assays:

  • Transfect enhancer-reporter constructs into relevant cell types

  • Use minimal promoters (e.g., SCP1) to isolate enhancer effects

  • Measure reporter gene expression under different conditions

  • Mutate specific transcription factor binding sites to identify crucial regulatory interactions

• CRISPR/Cas9-mediated enhancer deletion:

  • Generate enhancer knockout cell lines using CRISPR/Cas9

  • Differentiate cells into relevant lineages (e.g., endothelial cells)

  • Compare gene expression profiles between wildtype and enhancer-deleted cells

  • This approach revealed that deletion of enFLT1 resulted in 211 differentially expressed genes compared to wildtype cells

• Chromatin immunoprecipitation (ChIP):

  • Identify transcription factors binding to the enhancer in different tissues

  • The FLT1 enhancer (enFLT1) has been shown to be bound by multiple transcription factors involved in cardiovascular development, including NKX2-5

  • Compare binding patterns between normal and pathological conditions

• Chromosome conformation capture techniques:

  • Investigate long-range interactions between the enhancer and promoter

  • Determine how these interactions may differ across tissue types

  • Identify other genomic regions that may interact with the FLT1 enhancer

How should researchers address potential off-target effects when studying FLT1?

When studying FLT1 using gene silencing or editing approaches, researchers should implement these strategies to address off-target effects:

• For RNA interference experiments:

  • Use multiple siRNA sequences targeting different regions of FLT1

  • Include appropriate negative controls (non-targeting siRNA)

  • Verify specificity by confirming that expression of related receptors (e.g., KDR/VEGFR2) remains stable

  • Validate knockdown at both mRNA and protein levels

  • Published protocols have successfully demonstrated specific FLT1 knockdown without affecting KDR expression

• For CRISPR/Cas9 gene editing:

  • Use prediction tools to select guide RNAs with minimal off-target potential

  • Sequence multiple clones to confirm specific targeting

  • Perform whole-genome sequencing to identify potential off-target modifications

  • Include appropriate controls, such as targeting different regions of the gene

  • Create rescue experiments by reintroducing the deleted sequence

• Validation of phenotypes:

  • Confirm that observed phenotypes can be rescued by reintroduction of FLT1

  • Compare phenotypes between different knockdown/knockout strategies

  • Use multiple experimental approaches to validate findings

  • In studies of FLT1 enhancer deletion, comparing results with gene deletion helps distinguish enhancer-specific effects from general loss of function

• Cross-validation in different models:

  • Verify findings in both in vitro and in vivo systems when possible

  • Compare results between different cell types or model organisms

  • The human FLT1 enhancer function has been validated in both human cell lines and transgenic medaka models

What strategies can optimize detection of low-abundance FLT1 variants in experimental systems?

For optimal detection of low-abundance FLT1 variants, researchers should consider these methodological strategies:

• Sample preparation optimization:

  • Enrich for the cellular compartment where the variant is expected (membrane, cytosol, etc.)

  • Use appropriate extraction buffers to maximize protein recovery

  • Consider sample concentration techniques for low-abundance variants

• Western blot optimization:

  • Use high-sensitivity detection systems (e.g., chemiluminescent substrates)

  • Optimize antibody concentrations and incubation conditions

  • Consider non-reducing conditions for certain FLT1 variants

  • Use appropriate blocking reagents to minimize background

  • For FLT1-14 detection, non-reducing conditions and specific immunoblot buffer groups have been recommended

• PCR-based approaches:

  • Design primers specific to unique regions of FLT1 variants

  • Use nested PCR approaches for low-abundance transcripts

  • Consider digital PCR for absolute quantification of rare variants

  • Optimize annealing temperatures and cycle numbers

• Next-generation sequencing:

  • Use targeted sequencing approaches to increase coverage of FLT1 regions

  • Apply polyadenylation site sequencing (PAS-Seq) to identify alternative polyadenylation patterns

  • Implement RNA-Seq with sufficient depth to detect low-abundance transcripts

• Single-cell approaches:

  • Consider single-cell RNA-Seq to identify cell populations expressing specific variants

  • Use in situ hybridization techniques to localize variant expression in tissues

  • Employ immunofluorescence with variant-specific antibodies for protein localization

How might targeting FLT1 serve as a therapeutic strategy for preeclampsia or cardiovascular disease?

Several therapeutic strategies targeting FLT1 show promise for treating preeclampsia and cardiovascular diseases:

• RNA interference approaches:

  • The three predominant FLT1 mRNA isoforms represent potential targets for therapeutic RNAi in both early and late preeclampsia presentations

  • siRNA-based therapies could reduce excessive sFLT1 production in preeclampsia

  • Targeted delivery systems would be crucial to ensure placental specificity

• Enhancer-targeted therapies:

  • Modulating the activity of the FLT1 enhancer (enFLT1) could provide a more nuanced approach than targeting the gene itself

  • Enhancer-targeting compounds could fine-tune FLT1 expression without completely abolishing it

  • The 211 differentially expressed genes identified after enhancer deletion provide additional potential targets

• Anti-inflammatory approaches for CAD:

  • Since increased FLT1 expression due to genetic variants activates inflammatory cascades in CAD

  • Anti-inflammatory therapies targeting these pathways could benefit patients with FLT1-associated CAD risk

  • RNA interference experiments have shown that suppression of FLT1 decreases expression of inflammatory adhesion molecules

• sFLT1 neutralization:

  • Developing antibodies or decoy receptors that specifically bind sFLT1 without affecting membrane-bound FLT1

  • This approach could restore angiogenic balance in preeclampsia

  • Careful timing would be crucial to avoid interfering with normal placental development

• Personalized medicine approaches:

  • Genetic screening for FLT1 variants (e.g., rs74412485) could identify individuals at higher risk for CAD

  • These patients might benefit from earlier or more aggressive preventive interventions

  • Monitoring sFLT1 levels during pregnancy could help identify women at risk for preeclampsia before symptom onset

What are the most promising research frontiers in understanding FLT1 biology?

Several promising research frontiers are emerging in FLT1 biology:

• Comprehensive enhancer characterization:

  • Further investigation of the FLT1 enhancer network beyond enFLT1

  • Understanding tissue-specific enhancer activities and how they are integrated

  • Exploring the three-dimensional chromatin structure around the FLT1 locus

• Single-cell transcriptomics:

  • Characterizing FLT1 isoform expression at single-cell resolution in the placenta and other tissues

  • Identifying specific cell populations responsible for sFLT1 production in normal and pathological conditions

  • Mapping the cellular heterogeneity of FLT1 expression throughout development

• Systems biology approaches:

  • Integrating genomics, transcriptomics, and proteomics data to build comprehensive models of FLT1 regulation

  • Analyzing the 211 differentially expressed genes affected by enhancer deletion to identify key regulatory networks

  • Developing predictive models for how genetic variants affect FLT1 expression and function

• Evolutionary perspectives:

  • Exploring the primate-specific sFlt1-14 variant and its evolutionary significance

  • Comparing FLT1 enhancer function across species

  • Understanding why the human enhancer can drive gene expression in model organisms like medaka

• Precision medicine applications:

  • Developing clinical applications based on FLT1 genetic variants for CAD risk stratification

  • Creating predictive models for preeclampsia risk based on sFLT1 levels and genetic factors

  • Designing targeted therapies for specific patient populations based on FLT1-related biomarkers