FLT1 Human, His

What is the molecular structure of human FLT1 and how does the His-tag affect its functionality?

Human FLT1 is a tyrosine kinase receptor containing seven immunoglobulin-like domains in its extracellular region followed by a transmembrane domain and an intracellular tyrosine kinase domain. The gene consists of 30 exons and produces two main products: full-length FLT1 and a soluble form (sFLT1) derived from alternative splicing involving the first 13 exons with an intron-13-derived tail of 31 amino acids . The soluble form contains the first six Ig-like domains without the transmembrane or tyrosine kinase domains. When adding a His-tag for purification purposes, researchers should consider placing it at the N-terminus rather than the C-terminus to avoid interfering with signaling functions. Importantly, functionality assays comparing tagged versus untagged protein should be performed to ensure the tag does not disrupt binding to ligands such as VEGF-A.

What methods are effective for validating FLT1 RNA interference experiments?

When conducting RNA interference experiments targeting FLT1, validation requires multiple complementary approaches:

• mRNA quantification: Real-time PCR should show significant inhibition of FLT1 mRNA levels without affecting related receptors such as KDR (p<0.01 considered significant) .

• Protein verification: Western blotting using specific antibodies (e.g., monoclonal antibody against FLT1 at 1:500 dilution, V4262, Sigma-Aldrich) should confirm reduced protein expression (p<0.001 considered significant) .

• Specificity controls: Demonstrate no off-target effects on related receptors; for example, KDR expression should remain stable when FLT1 is knocked down and vice versa .

• Functional assays: Include tube formation assays, wound healing/migration assays, and proliferation tests to confirm biological effects of the knockdown .

For optimal transfection efficiency in primary human fetoplacental endothelial cells, researchers have successfully used commercially available siRNA targeting FLT1 with a recommended concentration of 20 nM .

How do FLT1 and KDR differentially regulate angiogenesis in fetoplacental endothelial cells?

FLT1 plays a dominant role in fetoplacental endothelial cell migration and tube formation compared to KDR, contradicting observations in other vascular beds where KDR is typically the primary mediator of angiogenesis. Research using RNA interference demonstrates that:

• FLT1 knockdown significantly impairs tube formation as early as 2 hours after plating, with maximum impairment at 12 hours post-plating. Quantitatively, FLT1-ablated cells show significantly decreased branch points (p<0.001) and total tube length (p<0.001) .

• KDR knockdown, in contrast, does not significantly affect tube formation at any time point, suggesting a secondary role in fetoplacental angiogenesis .

• FLT1 appears to mediate migration-dependent processes rather than proliferation in these cells, as evidenced by wound scratch assays .

This differential regulation suggests that therapeutic approaches targeting angiogenesis in placental disorders should prioritize FLT1-mediated pathways rather than KDR-mediated pathways, contrary to approaches used in cancer angiogenesis.

What is the relationship between FLT1 genetic variants and longevity, particularly in the context of hypertension?

FLT1 genetic variants show a significant association with longevity, particularly through interactions with hypertension status. The SNP rs3794396 exhibits the most statistically significant association with longevity (p=0.0007) . Research data demonstrate:

Model 1 is adjusted for age, while Model 2 is adjusted for additional factors including BMI, glucose levels, and lifestyle factors .

The data reveal that in hypertensive individuals, the GG genotype confers a 12% lower mortality risk compared to GC/CC genotypes, while individuals with GG or CC genotypes have a 22% lower mortality risk compared to those with the GC genotype . This protective effect is not observed in normotensive individuals, suggesting that FLT1 genetic variants specifically modulate the mortality risk associated with hypertension.

How can the human FLT1 regulatory element (enFLT1) be effectively studied in developmental models?

The human FLT1 regulatory element (enFLT1) is a critical cis-regulatory element (CRE) involved in cardiovascular development. To effectively study this element:

When designing these experiments, researchers should consider evolutionary conservation of the enhancer sequence as an indicator of functional importance and validate findings across multiple model systems.

What are the optimal protein extraction and detection methods for studying FLT1 in primary human endothelial cells?

For studying FLT1 in primary human endothelial cells, the following methodological approach has been validated:

• Protein extraction: Use M-PER Mammalian Protein Extraction Reagent (Thermo Fisher) supplemented with phosphatase and protease inhibitors (Cell Signaling Technologies) . Extract supernatant by centrifugation and determine protein concentration via BCA protein assay.

• SDS-PAGE resolution: Load 10 μg of whole cell lysate on 4-15% precise gels before transferring to polyvinylidene difluoride (PVDF) membranes .

• Antibody selection and dilution:

  • Primary antibody: Monoclonal antibody against FLT1 (1:500, V4262, Sigma-Aldrich)

  • Loading control: Monoclonal antibody anti-β-actin (1:2000, A1978, Sigma-Aldrich)

• Signaling studies: For investigating VEGFA-stimulated signaling, starve cells for 1 hour after siRNA transfection, then treat with VEGFA (R&D, 293-VE) at 60ng/ml for varying time points (0, 5, 10, and 60 min) in serum- and growth factor-free medium .

• Downstream signaling detection: For pathway analysis, use the following antibodies:

  • Phospho-Akt (1:1000, 9271S, Cell Signaling Technology)

  • Total Akt (1:1000, 9272S, Cell Signaling Technology)

  • Phospho-Erk (1:1000, 9101S, Cell Signaling Technology)

  • Total Erk (1:2000, 4696S, Cell Signaling Technology)

This methodology allows for reliable quantification of both FLT1 protein levels and its signaling effects in primary human endothelial cells.

What experimental designs effectively distinguish between FLT1 and sFLT1 functions in pathological conditions?

To effectively distinguish between the functions of full-length FLT1 and soluble FLT1 (sFLT1) in pathological conditions, researchers should implement the following experimental design strategies:

• Genetic models: Utilize Flt-1 TK−/− mice, which are defective only in FLT1 signaling while maintaining the VEGF-trapping function of sFLT1. These models have been successfully used to demonstrate slower growth of transplanted cancer cells and reduced metastasis compared to wild-type mice, highlighting the role of FLT1 signaling in tumor progression .

• Specific inhibition: Employ RNA interference with sequence-specific siRNAs targeting either:

  • Common regions (affects both forms)

  • Transmembrane/intracellular domains (affects only full-length FLT1)

  • The unique 31-amino acid tail of sFLT1

• Functional assays: Apply tube formation, wound scratch, and MTT assays after specific knockdown to assess the individual contributions of each form to angiogenic processes .

• Plasma level measurements: In conditions such as preeclampsia, measure plasma sFLT1 levels at different time points to establish temporal relationships between sFLT1 elevation and symptom onset. Studies have shown that patients affected by preeclampsia have increased plasma sFLT1 levels early in pregnancy, before detectable signs of the condition appear .

• Complementation experiments: Perform rescue experiments with recombinant proteins or expression constructs of either full-length FLT1 or sFLT1 following knockdown of the endogenous proteins to distinguish their specific functions.

How can researchers effectively analyze the interaction between FLT1 genotypes and disease states in longitudinal studies?

For analyzing interactions between FLT1 genotypes and disease states in longitudinal studies, researchers should follow these methodological approaches:

• Genotyping strategy: Select tagging SNPs (tSNPs) that capture most of the genetic variation in FLT1, including 5 kb of the 5' and 3' flanking DNA. Target SNPs with significant associations after correction for multiple testing, such as rs3794396 which has shown strong association with longevity (p=0.0007) .

• Statistical models: Test multiple genetic models to identify the most significant associations:

  • Major allele recessive model (GG vs. other genotypes)

  • Heterozygote disadvantage model (GC vs. other genotypes)

  • Minor allele homozygote model (CC vs. other genotypes)

  • Additive model (effect by number of C alleles)

• Interaction analysis: Employ Cox proportional hazards models with interaction terms to assess how FLT1 variants modify disease effects on mortality. Include adjustment for relevant covariates such as:

  • Age

  • BMI

  • Fasting glucose

  • Smoking (pack-years)

  • Alcohol intake

  • Physical activity index

  • Comorbid conditions

• Stratified analysis: Analyze data separately for subjects with and without specific conditions (e.g., hypertension vs. normotension) to identify condition-specific effects of genotypes, as shown in this interaction effect table:

• Survival analysis: Generate Kaplan-Meier survival curves for each genotype stratified by disease state to visually represent differential effects of genotypes on survival .

How can the dual roles of FLT1 in angiogenesis be reconciled for therapeutic targeting?

FLT1 exhibits a fascinating dual role in angiogenesis: a negative role in early embryogenesis and a positive role in pathological conditions like cancer. To reconcile these opposing functions for therapeutic targeting:

• Context-specific signaling: Investigate the molecular switches that determine whether FLT1 acts as a positive or negative regulator in different tissues or disease states. This includes identifying specific co-receptors, ligand concentrations, and downstream signaling modulators .

• Domain-specific targeting: Design therapeutic agents that specifically target domains responsible for pathological activation while preserving physiological functions. This requires detailed structural and functional mapping of FLT1 domains involved in different signaling cascades .

• Temporal targeting strategies: Develop temporally controlled therapeutic interventions that account for the developmental stage-specific roles of FLT1. For example, VEGF-VEGFR inhibitors used in cancer treatment cause hypertension and proteinuria, reminiscent of preeclampsia symptoms, highlighting the importance of understanding temporal aspects of FLT1 function .

• Cell-type specific approaches: Distinguish between FLT1 functions in different cell types, as FLT1 signaling in macrophages may have different implications than in endothelial cells. Studies have shown that wild-type mice carrying Flt-1 TK−/− bone marrow exhibit slower growth of transplanted tumors, suggesting an important role for FLT1 in bone marrow-derived cells .

What are the methodological considerations for studying the enFLT1 enhancer's role in cardiovascular development disorders?

When studying the enFLT1 enhancer in cardiovascular development disorders, researchers should consider these methodological approaches:

• Evolutionary conservation analysis: Prioritize highly conserved regions within the enhancer, as this conservation suggests functional importance across species. The human enFLT1 has demonstrated conserved function in driving reporter expression in the cardiovascular system of model organisms .

• CRISPR-based enhancer editing: Rather than complete deletion, consider designing precise edits that modify specific transcription factor binding sites within the enhancer to dissect its functional components. This approach can reveal which aspects of the enhancer contribute to different developmental processes .

• Single-cell transcriptomics: Apply single-cell RNA sequencing before and after enhancer manipulation to identify cell-type specific effects on gene expression programs that might be masked in bulk tissue analysis .

• 4D genome analysis: Employ chromosome conformation capture (Hi-C or similar) techniques to understand the three-dimensional interactions between enFLT1 and its target promoters across developmental time points .

• Multi-species validation: Test enhancer function across multiple model systems (zebrafish, medaka, mouse) to distinguish conserved functions from species-specific roles, particularly important given the differences in FLT1 gene structure across vertebrates .