VCAM1 Human, HEK

What is VCAM1 and what are its primary functions in human vascular biology?

VCAM1 (Vascular Cell Adhesion Molecule-1) is a critical glycoprotein expressed primarily on activated endothelial cells during inflammatory responses. It functions as an adhesion molecule that mediates leukocyte-endothelial cell interactions by binding to integrin α4β1 (VLA-4) on leukocytes. In vascular biology, VCAM1 plays essential roles in mediating immune cell recruitment to sites of inflammation, facilitating intercellular communication through extracellular vesicles (EVs), and contributing to vascular homeostasis. Recent research has demonstrated that VCAM1 is not only expressed on endothelial cells but also on hematopoietic stem cells (HSCs) where it serves as a quality-control checkpoint for bone marrow entry by providing a "don't-eat-me" signal in the context of MHC class-I presentation . This multifaceted role makes VCAM1 a significant molecule in both normal physiology and pathological conditions like cardiovascular disease.

How is VCAM1 expression induced and regulated in human endothelial cells?

VCAM1 expression in human endothelial cells is primarily induced by pro-inflammatory cytokines, particularly TNF-α. When human umbilical vein endothelial cells (HUVECs) are exposed to TNF-α, they exhibit a significant increase in VCAM1 expression at both the mRNA and protein levels. Studies have shown that TNF-α stimulation of HUVECs leads to elevated VCAM1 protein levels in cell culture supernatants within 6-18 hours (P < 0.001 compared to control conditions) . This upregulation occurs through activation of NF-κB signaling pathways. The regulation of VCAM1 involves transcriptional control, post-translational modifications, and membrane trafficking processes. Additionally, epigenetic mechanisms and microRNAs can modulate VCAM1 expression under different physiological and pathological conditions. Understanding these regulatory mechanisms is crucial for designing experiments that accurately assess VCAM1 function in HEK293 expression systems.

What methodological approaches are most effective for studying VCAM1 in experimental settings?

For studying VCAM1 in experimental settings, researchers should consider a multi-faceted approach including:

• Gene expression analysis: Digital droplet PCR (ddPCR) has proven highly effective for quantifying VCAM1 mRNA levels with high sensitivity. This technique has successfully demonstrated differential expression of VCAM1 and related adhesion molecules like ICAM-1 in TNF-α stimulated versus control HUVECs (P < 0.001) .

• Protein detection: ELISA assays can reliably measure soluble VCAM1 in cell culture supernatants, while immunoblotting techniques can assess membrane-bound forms.

• Functional analysis: The VCAM1-EVHB-Chip microfluidic system provides an innovative approach for capturing VCAM1+ cells and VCAM1+ extracellular vesicles with high specificity. This system has been validated using both engineered endothelial cells overexpressing VCAM1 and TNF-α-stimulated HUVECs .

• Cellular imaging: Fluorescence microscopy with appropriate nuclear staining (e.g., DAPI) can visualize VCAM1+ cells captured on specialized surfaces. Widefield fluorescence microscopy using a 60X ELWD PlanApomat objective has been successfully employed for this purpose .

Each of these methodologies offers distinct advantages depending on the specific research question being addressed.

What are the optimal conditions for expressing human VCAM1 in HEK293 cells?

For optimal expression of human VCAM1 in HEK293 cells, researchers should consider the following protocol based on recent successful approaches:

• Vector selection: Use a mammalian expression vector containing a strong promoter (e.g., CMV) and appropriate selection marker. For visualization and tracking, consider fusion constructs such as TdTomato-palmitoylation-VCAM1, which has been successfully expressed in endothelial cell lines .

• Transfection conditions: Lipid-based transfection methods typically yield good results with HEK293 cells. Maintain cells at 70-80% confluence in complete DMEM medium supplemented with 10% FBS at transfection.

• Post-transfection culture: After transfection, culture cells for 24-48 hours before assessing expression. Addition of TNF-α (10 ng/mL) for 6-18 hours can enhance VCAM1 expression if mimicking inflammatory conditions is desired.

• Selection and maintenance: For stable expression, apply appropriate selection pressure and validate expression through multiple passages.

• Expression validation: Confirm VCAM1 expression using flow cytometry, ELISA of culture supernatants for soluble VCAM1, and Western blotting for cell-associated VCAM1. Successful expression should yield significantly higher VCAM1 levels compared to non-transfected controls (P < 0.001) .

These optimized conditions ensure robust and reproducible VCAM1 expression in the HEK293 cellular background.

How can I validate successful expression and functionality of human VCAM1 in HEK293 cells?

Validating both the expression and functionality of human VCAM1 in HEK293 cells requires multiple complementary approaches:

• Expression validation:

  • Western blot analysis: Using anti-VCAM1 antibodies to detect the protein at the expected molecular weight.

  • Flow cytometry: Using fluorescently-labeled anti-VCAM1 antibodies to quantify surface expression.

  • ELISA: Measuring soluble VCAM1 in culture supernatants, which should show significant elevation compared to control cells (P < 0.001) .

• Functional validation:

  • Adhesion assays: Testing the ability of VCAM1-expressing HEK293 cells to bind to VLA-4 expressing cells or recombinant VLA-4.

  • Microfluidic capture: Using a VCAM1-EVHB-Chip to capture VCAM1-expressing cells. VCAM1-positive cells should show robust binding to anti-VCAM1 coated surfaces but minimal binding to IgG control surfaces .

  • Extracellular vesicle analysis: Isolating EVs from culture media and confirming VCAM1 presence using Western blot or specialized EV protein arrays to detect markers such as CD9, CD63, HSP70, and ALIX alongside VCAM1 .

• Activation response: If mimicking inflammatory conditions, validate that VCAM1 expression increases in response to TNF-α treatment, similar to what occurs in endothelial cells.

These validation steps ensure that the expressed VCAM1 is not only present but also correctly folded and functional in your experimental system.

What challenges might I encounter when expressing human VCAM1 in HEK293 cells and how can I address them?

When expressing human VCAM1 in HEK293 cells, researchers may encounter several challenges:

• Variable expression levels: HEK293 cells can exhibit heterogeneous expression levels of transfected proteins. To address this:

  • Use FACS to isolate high-expressing populations

  • Consider inducible expression systems for better control

  • Optimize codon usage for human expression

• Post-translational modifications: HEK293 cells may not perfectly recapitulate the glycosylation pattern of VCAM1 seen in endothelial cells. If glycosylation is critical for your study:

  • Validate glycosylation patterns using specific glycosidases

  • Consider enzymatic deglycosylation followed by Western blotting to compare with native VCAM1

• Cellular localization issues: VCAM1 may not properly localize to the cell membrane. Solutions include:

  • Using a palmitoylation tag to enhance membrane targeting (as demonstrated with TdTomato-palmitoylation-VCAM1 constructs)

  • Employing immunofluorescence microscopy to confirm proper membrane localization

• Functional validation difficulties: Expressed VCAM1 may be present but not fully functional. Address this by:

  • Performing adhesion assays with VLA-4 expressing cells

  • Using the VCAM1-EVHB-Chip to confirm binding capacity

  • Testing the ability of expressed VCAM1 to activate relevant signaling pathways

By anticipating and addressing these challenges, researchers can optimize their VCAM1 expression systems for more reliable experimental outcomes.

How can microfluidic technology be utilized to isolate and study VCAM1+ cells and extracellular vesicles?

Microfluidic technology offers a powerful approach for isolating and studying VCAM1+ cells and extracellular vesicles, particularly through the VCAM1-EVHB-Chip (Extracellular Vesicles Herringbone Chip) system. This innovative methodology addresses the challenges of isolating specific subpopulations of extracellular vesicles that express VCAM1, which has traditionally been difficult using conventional techniques.

The VCAM1-EVHB-Chip functions through the following principles:

• Device design: The herringbone-patterned microfluidic chip creates microvortices that enhance interactions between target cells/EVs and the antibody-coated surface .

• Surface functionalization: The chip surface is coated with anti-VCAM1 antibodies via aryl diazonium fabrication of plastic and nanoparticle-based modifications, providing high specificity for VCAM1+ entities .

• Capture protocol: For EV capture, 500 μL of concentrated conditioned media is flowed through each device at a rate of 1 mL/h, followed by washing with 1.5 mL of PBS at 1.5 mL/h .

• Visualization: The thin TOPAZ layer (170 μm) sealing the device allows direct imaging of captured cells and EVs using fluorescence microscopy .

This system has demonstrated selective capture of both VCAM1+ cells (such as TNF-α-stimulated HUVECs) and VCAM1+ extracellular vesicles, allowing subsequent analysis of their molecular cargo. Research has shown significant enrichment of specific mRNAs (such as ICAM-1, P < 0.001) in captured VCAM1+ EVs, providing insights into their potential biological functions .

What is the role of VCAM1 in hematopoietic stem cell engraftment and immune tolerance?

VCAM1 plays a critical and previously underappreciated role in hematopoietic stem cell (HSC) engraftment and immune tolerance. Recent research has revealed that VCAM1 is expressed on healthy HSCs and upregulated on leukemic stem cells (LSCs), where it functions as a quality-control checkpoint for bone marrow entry .

Key findings regarding VCAM1's role in HSC engraftment include:

• "Don't-eat-me" signaling: VCAM1 provides a protective signal in the context of MHC class-I presentation, preventing phagocytic clearance of HSCs during engraftment .

• MHC mismatch tolerance: While haplotype-matched HSCs can engraft regardless of VCAM1 status, VCAM1 deletion leads to striking defects in engraftment specifically in the context of haplotype-mismatched transplantation. Experiments with haplotype-mismatched VCAM1-deficient cells resulted in failed engraftment and approximately 76% mortality in recipients .

• Phagocyte interaction: Gr1^low monocytes were identified as the most active cellular population in clearing VCAM1-deficient cells, indicating that VCAM1 specifically protects against clearance by this phagocytic subset .

These findings have significant implications for both normal hematopoiesis and malignant conditions like acute myeloid leukemia (AML), suggesting that VCAM1 expression serves as a crucial immune evasion mechanism that can be exploited by cancer stem cells.

How does VCAM1 expression affect outcomes in acute myeloid leukemia (AML)?

VCAM1 expression has significant impacts on the progression and outcomes of acute myeloid leukemia (AML). Analysis of patient data has revealed a correlation between high VCAM1 expression and reduced survival in AML patients . These clinical observations have been substantiated by experimental studies exploring the mechanistic basis of VCAM1's role in AML progression:

• Disease progression: When VCAM1 was overexpressed in MOLM-13 AML cells (which are naturally VCAM1 negative) and transplanted into NSG mice, these cells showed significantly more rapid disease progression compared to control MOLM-13 cells .

• Survival impact: Mice receiving hVCAM1-MOLM-13 cells exhibited significantly reduced survival compared to those receiving control MOLM-13 cells, demonstrating VCAM1's role in accelerating disease progression .

• Therapeutic targeting: Administration of anti-human VCAM1 antibodies significantly extended the survival of mice transplanted with hVCAM1-MOLM-13 cells, whereas targeting mouse VCAM1 in the recipient microenvironment did not affect outcomes . This finding highlights the specific contribution of leukemic cell-expressed VCAM1 to disease progression.

• Mechanism independence: The aggressive phenotype of VCAM1-expressing AML cells was not attributable to differences in homing capacity, cell viability, or cell cycling, suggesting other immune evasion mechanisms at play .

These findings indicate that VCAM1 expression in AML serves as both a prognostic indicator and a potential therapeutic target, particularly through its role in mediating immune evasion.

What methods can be used to study VCAM1+ extracellular vesicles in cardiovascular research?

Studying VCAM1+ extracellular vesicles (EVs) in cardiovascular research requires specialized methodologies to isolate, characterize, and analyze these important signaling entities. The following approaches have proven effective:

• Isolation techniques:

  • Microfluidic capture: The VCAM1-EVHB-Chip provides selective isolation of VCAM1+ EVs through antibody-coated surfaces in a herringbone-patterned microfluidic device .

  • Differential ultracentrifugation: Can be used for initial EV enrichment before specific VCAM1+ EV isolation.

  • Immunoaffinity purification: Using anti-VCAM1 antibodies conjugated to magnetic beads or other solid supports.

• Characterization methods:

  • Size determination: Nanoparticle Tracking Analysis has successfully characterized HUVEC-derived EC-EVs, revealing a modal size of 83.5 nm .

  • Marker analysis: Western blotting for EV markers (CD9, CD63, HSP70, ALIX) alongside VCAM1 verification .

  • Molecular cargo analysis: Digital droplet PCR (ddPCR) for precise quantification of mRNA transcripts within isolated VCAM1+ EVs .

• Functional assessment:

  • In vitro cell treatment: Applying isolated VCAM1+ EVs to recipient cells (e.g., immune cells, endothelial cells) to assess functional responses.

  • In vivo modeling: Administering labeled VCAM1+ EVs to animal models of cardiovascular disease to track biodistribution and effects.

• Inflammatory activation models:

  • TNF-α stimulation of endothelial cells (e.g., HUVECs) has been established as an effective method to increase VCAM1+ EV production for subsequent study .

These methodologies enable researchers to investigate the role of VCAM1+ EVs in intercellular signaling during vascular inflammation, immune cell recruitment, and the progression of cardiovascular diseases.

How can I address low detection of VCAM1 in experimental systems?

When facing challenges with low VCAM1 detection in experimental systems, consider the following troubleshooting approaches:

• Expression optimization:

  • Stimulus intensity: If using TNF-α to induce VCAM1 expression, optimize both concentration (typically 10-20 ng/mL) and duration (6-18 hours have shown significant increases in VCAM1 levels, P < 0.001) .

  • Vector design: Ensure your expression construct contains optimal Kozak sequences and lacks inhibitory secondary structures in the 5' UTR.

  • Promoter selection: Consider stronger promoters or inducible systems for better control of expression timing.

• Detection optimization:

  • Antibody selection: Test multiple anti-VCAM1 antibody clones as epitope accessibility may vary depending on protein conformation and post-translational modifications.

  • Signal amplification: For Western blotting, consider enhanced chemiluminescence substrates; for ELISA, explore biotin-streptavidin amplification systems.

  • Sample preparation: For membrane-bound VCAM1, optimize lysis conditions to effectively solubilize membrane proteins without denaturing epitopes.

• Extracellular vesicle isolation enhancement:

  • For VCAM1+ EVs, concentrate conditioned media before flowing through the VCAM1-EVHB-Chip to increase capture efficiency .

  • Optimize flow rates (1 mL/h has been effective) and washing protocols (1.5 mL PBS at 1.5 mL/h) to maximize specific binding while minimizing background .

• Analytical sensitivity improvement:

  • Use digital droplet PCR instead of conventional qPCR for more sensitive and absolute quantification of VCAM1 transcripts .

  • Consider mass spectrometry-based approaches for protein detection if antibody-based methods prove insufficient.

These strategies should help address most detection challenges encountered in VCAM1 research systems.

What controls should be included when studying VCAM1 in experimental settings?

Robust experimental design for VCAM1 studies requires carefully selected controls to ensure valid interpretation of results:

• Expression controls:

  • Positive control: Include a cell line known to express high levels of VCAM1 (e.g., TNF-α-stimulated HUVECs) .

  • Negative control: Use matched cell lines without VCAM1 expression or with VCAM1 knockdown/knockout.

  • Induction control: For TNF-α stimulation experiments, include time-matched unstimulated cells to account for time-dependent changes unrelated to VCAM1 induction .

• Antibody controls:

  • Isotype control: Use matched isotype antibodies (e.g., IgG controls for anti-VCAM1 antibodies) in flow cytometry, immunoprecipitation, or microfluidic capture experiments .

  • Peptide competition: To confirm antibody specificity, pre-incubate anti-VCAM1 antibodies with excess recombinant VCAM1 or specific peptides.

• Microfluidic device controls:

  • For VCAM1-EVHB-Chip experiments, parallel IgG-coated devices serve as critical specificity controls .

  • Flow known VCAM1-negative EVs or cells through the VCAM1-coated device to establish background binding levels.

• Extracellular vesicle controls:

  • Verify EV identity by confirming the presence of established EV markers (CD9, CD63, HSP70, ALIX) alongside VCAM1 .

  • Include size characterization via Nanoparticle Tracking Analysis to confirm typical EV size distribution (e.g., modal size of 83.5 nm for HUVEC-derived EVs) .

• PCR controls:

  • Include housekeeping genes for normalization in gene expression studies.

  • For digital droplet PCR, include no-template controls and positive controls with known copy numbers.

Proper implementation of these controls ensures experimental rigor and facilitates accurate interpretation of VCAM1-related findings.

How can I analyze complex VCAM1+ extracellular vesicle data from microfluidic isolation systems?

Analyzing complex data from VCAM1+ extracellular vesicle (EV) isolation using microfluidic systems requires a systematic approach:

• Capture efficiency assessment:

  • Compare EV yields between VCAM1-EVHB-Chip and IgG control devices to determine specific versus non-specific binding .

  • Quantify capture using EV marker proteins (CD9, CD63, HSP70, ALIX) via Western blotting or specialized EV protein arrays .

• Cargo analysis workflows:

  • Transcript quantification: Digital droplet PCR (ddPCR) provides absolute quantification of specific transcripts within VCAM1+ EVs. Statistical analysis should compare target genes between experimental conditions using appropriate statistical tests (e.g., Student's t-test for comparing TNF-α stimulated versus control samples) .

  • Protein profiling: Mass spectrometry-based proteomics can provide comprehensive protein profiles of captured EVs.

  • RNA sequencing: For global transcriptome analysis, implement specialized small RNA sequencing protocols optimized for EV RNA content.

• Data normalization approaches:

  • For RNA analyses, normalize to established EV RNA markers or spike-in controls rather than cellular housekeeping genes.

  • For protein analyses, normalize to EV markers (CD9, CD63) or total protein content.

• Visualization and statistical analysis:

  • Present data as means ± standard deviation from multiple independent experiments (e.g., three independent experiments per group as in published studies) .

  • Apply appropriate statistical tests (Student's t-test for two-group comparisons) and clearly denote significance levels (e.g., **** P < 0.0001) .

  • Create comprehensive visualization of molecular signatures using heatmaps or volcano plots to identify differentially expressed genes or proteins.

• Functional correlation:

  • Correlate molecular cargo profiles with functional outcomes in recipient cells or disease models to establish biological relevance.

This structured analytical approach allows researchers to extract meaningful insights from complex VCAM1+ EV data generated using microfluidic isolation systems.

How might VCAM1-targeted approaches be developed for therapeutic applications?

VCAM1-targeted therapeutic approaches represent a promising frontier in treating cardiovascular, inflammatory, and oncological conditions. Several strategic directions warrant exploration:

• AML treatment strategies:

  • Anti-VCAM1 antibody therapy has shown significant promise in extending survival in AML models. Experiments demonstrated that administration of anti-human VCAM1 antibodies significantly extended survival in mice transplanted with VCAM1-expressing MOLM-13 AML cells .

  • Combinatorial approaches pairing VCAM1 targeting with conventional chemotherapeutics could enhance efficacy by simultaneously addressing immune evasion and proliferation.

• Cardiovascular disease interventions:

  • Targeting VCAM1+ endothelial extracellular vesicles could provide a novel approach to modulating vascular inflammation without broadly suppressing immune function.

  • Development of small molecule inhibitors that disrupt VCAM1-VLA-4 interactions specifically in pathological contexts could offer therapeutic benefits with minimal side effects.

• Delivery system innovations:

  • Adapting the VCAM1-EVHB-Chip technology from diagnostic to therapeutic applications could enable selective removal of VCAM1+ cells or EVs from circulation in conditions where they contribute to pathology .

  • Engineering therapeutic EVs that target VCAM1-expressing cells could provide precise delivery of therapeutic cargo to sites of inflammation or malignancy.

• Hematopoietic stem cell transplantation:

  • Manipulating VCAM1 expression on donor HSCs could enhance engraftment in haplotype-mismatched transplantation scenarios, potentially expanding the donor pool for patients requiring transplantation .

  • Temporary VCAM1 upregulation during the engraftment window, followed by normalization, might optimize outcomes.

These approaches represent promising avenues for translating fundamental VCAM1 biology into clinical applications, though each requires extensive preclinical validation before human translation.

What emerging technologies might advance our understanding of VCAM1 biology in human systems?

Several cutting-edge technologies are poised to significantly advance our understanding of VCAM1 biology in human systems:

• Next-generation microfluidic platforms:

  • Building upon the VCAM1-EVHB-Chip, next-generation devices could integrate capture, analysis, and functional testing of VCAM1+ cells and EVs in a single platform .

  • Multiparametric microfluidic systems could simultaneously isolate and characterize multiple EV subpopulations based on VCAM1 co-expression with other markers.

• Single-EV analysis technologies:

  • Flow nanometry and similar technologies enable analysis of individual EVs rather than bulk populations, potentially revealing heterogeneity within VCAM1+ EV populations.

  • Coupling single-EV isolation with molecular barcoding could allow comprehensive characterization of cargo at unprecedented resolution.

• Advanced imaging approaches:

  • Super-resolution microscopy techniques could visualize VCAM1 distribution and clustering at the nanoscale, providing insights into its functional organization.

  • Intravital imaging of fluorescently labeled VCAM1+ EVs could track their biodistribution and cellular interactions in real-time within living organisms.

• CRISPR-based functional genomics:

  • CRISPR activation/interference screens could systematically identify regulators of VCAM1 expression and function in various cellular contexts.

  • Base editing or prime editing approaches could enable precise modification of VCAM1 to dissect structure-function relationships.

• Artificial intelligence integration:

  • Machine learning algorithms applied to large datasets of VCAM1 expression patterns across diseases could identify novel associations and therapeutic opportunities.

  • Predictive modeling could optimize experimental design for VCAM1 studies and accelerate therapeutic development.

By leveraging these emerging technologies, researchers can gain unprecedented insights into VCAM1 biology, potentially revealing new roles in health and disease beyond current understanding.

How does VCAM1 expression on extracellular vesicles influence intercellular communication in disease states?

VCAM1 expression on extracellular vesicles (EVs) represents a sophisticated mechanism of intercellular communication with significant implications for disease pathogenesis. Current research suggests several key effects of VCAM1+ EVs on recipient cells and tissues:

• Targeted cellular communication:

  • VCAM1+ EVs can selectively interact with cells expressing VLA-4 (α4β1 integrin), providing a mechanism for targeted delivery of EV cargo to specific cell populations.

  • This targeting capability enables VCAM1+ EC-EVs to mediate signaling between endothelial cells and immune cells during vascular inflammation .

• Transcriptional reprogramming:

  • Analysis of VCAM1+ EC-EVs has revealed enrichment of specific mRNA transcripts, particularly ICAM-1 (P < 0.001) . This specialized cargo suggests that VCAM1+ EVs may transfer specific molecular information that reprograms recipient cell function.

  • The transfer of these transcripts could induce pro-inflammatory phenotypes in recipient cells, potentially amplifying inflammatory responses in cardiovascular and metabolic diseases.

• Immune cell deployment and recruitment:

  • VCAM1+ EC-EVs have been implicated in signaling the deployment of immune cells from splenic reserves during cardiovascular disease . This mechanism represents a long-distance communication pathway between damaged vasculature and immune system mobilization.

  • The specific interaction between VCAM1 on EVs and immune cell integrins may facilitate precise recruitment patterns that contribute to disease progression.

• Pathological intercellular signaling:

  • In the context of acute myeloid leukemia, elevated VCAM1 expression correlates with reduced patient survival . VCAM1+ EVs may contribute to this poor prognosis by transmitting signals that promote disease progression or therapy resistance.

Future research utilizing the VCAM1-EVHB-Chip and other advanced technologies will likely uncover additional mechanisms through which VCAM1+ EVs influence disease progression, potentially revealing new therapeutic targets for intervention .