VASH1 Antibody

What is VASH1 and what are its primary functions in biological systems?

VASH1 (Vasohibin-1) is a novel angiogenic regulatory factor that belongs to the vasohibin family of proteins. Located on human chromosome 14q24, VASH1 primarily functions as an endogenous angiogenesis inhibitor but also serves several other important biological roles. Unlike typical angiogenesis inhibitors that induce endothelial cell death, VASH1 enhances endothelial cell maintenance by strengthening stress resistance through upregulation of superoxide dismutase 2 (which quenches reactive oxygen species) and synthesis of Sirtuin 1 (SIRT1), an anti-aging protein .

VASH1 demonstrates multiple biological functions beyond angiogenesis regulation, including:

• Inhibition of tumor growth and metastasis in various cancer models

• Promotion of microtubule formation affecting cell migration and invasion

• Regulation of immune cell infiltration and immune microenvironment remodeling

This multifaceted role makes VASH1 an important target for research across various fields including cancer biology, vascular biology, and immunology.

How does VASH1 expression vary across different cell types and tissues?

VASH1 expression is more extensive than initially thought. While primarily expressed in endothelial cells, research has demonstrated a broader distribution pattern:

• Endothelial cells: Principal site of VASH1 expression

• Vascular smooth muscle cells: Show weak expression that can be induced by platelet-derived growth factors

• Fibroblasts: Express very low levels, generally unresponsive to FGF-2 stimulation

• Brain, heart, and kidney: Weak expression of VASH1 mRNA detected

• Immune cells: Detected in monocyte-derived macrophages and peripheral blood mononuclear cells

• Muscle tissue: Present in cardiac myocytes in vitro and striated muscles of adult rats

• Tumor cells: Variable expression across different cancer types

This extensive expression profile suggests that VASH1 may have broader physiological functions beyond its initially described role in angiogenesis regulation. Understanding this expression pattern is crucial when designing experiments with VASH1 antibodies to ensure appropriate positive and negative controls.

What are the key differences between VASH1 isoforms and how do they affect antibody selection?

VASH1 exists in multiple isoforms, with VASH1A and VASH1B being the most well-characterized. These isoforms exhibit distinct biological properties that can significantly impact antibody selection and experimental design:

VASH1A:

• Full-length protein with moderate effects on endothelial cell behavior

• Contains all exons of the VASH1 gene

VASH1B:

• Lacks exons 6-8 due to alternative splicing

• More potently inhibits endothelial cell growth, migration, and capillary formation

• Induces apoptosis in proliferating human fibroblasts and cancer cells

When selecting VASH1 antibodies, researchers should consider:

• Whether the antibody can distinguish between VASH1A and VASH1B

• The epitope location relative to the spliced regions in VASH1B

• The experimental goal (detecting total VASH1 vs. specific isoforms)

• The potential for cross-reactivity with VASH2, another vasohibin family member

For studies focusing on isoform-specific functions, antibodies targeting unique epitopes created by alternative splicing are essential for accurate experimental outcomes.

How is VASH1 expression associated with cancer prognosis and what methodologies are used to evaluate this?

VASH1 expression shows variable prognostic significance across different cancer types, requiring careful methodological approaches for accurate evaluation:

In Lower-Grade Glioma (LGG):

• High VASH1 expression correlates with poor prognosis (HR = 4.753, P=0.002)

• Associated with higher WHO grade, IDH1 wild-type status, and progressive disease

In other cancers:

• Ovarian cancer: Silenced VASH1 expression worsens prognosis

• Colon cancer: Different isoforms show distinct effects (VASH1-A induces senescence; VASH1-B induces apoptosis)

Methodological approaches for evaluating VASH1 as a prognostic marker include:

• Immunohistochemical analysis with validated VASH1 antibodies

• Multivariate Cox regression analysis to account for confounding factors

• Nomogram construction incorporating VASH1 with other established prognostic markers

• Validation using C-index and AUC curve analysis

• Patient stratification by clinical characteristics to assess consistency across subgroups

Researchers should employ multiple complementary techniques when evaluating VASH1's prognostic significance to account for its context-dependent roles in different tumor types.

What experimental approaches can be used to study VASH1 function in tumor angiogenesis?

Studying VASH1's role in tumor angiogenesis requires multifaceted experimental approaches:

• In vitro angiogenesis assays with VASH1 antibody applications:

  • Tube formation assay: Determine how VASH1 affects endothelial network formation

  • Wound healing assay: Assess VASH1's impact on endothelial cell migration

  • Spheroid sprouting assay: Evaluate three-dimensional angiogenic responses

• VASH1 knockdown/overexpression studies:

  • Generate stable VASH1 knockdown cell lines using shRNA (e.g., in U-251 glioma cells)

  • Create VASH1-overexpressing cells to study dose-dependent effects

  • Validate knockdown efficiency using both qRT-PCR and Western blot with VASH1 antibodies

• Co-culture systems:

  • Culture tumor cells with endothelial cells to study paracrine effects

  • Use VASH1 antibodies in immunofluorescence to track protein localization

• In vivo angiogenesis models:

  • Chorioallantoic membrane (CAM) assay with VASH1 protein or expressing cells

  • Matrigel plug assay with VASH1 manipulation

  • Tumor xenografts with VASH1-modified cells

• Immunohistochemical analysis of tumor vasculature:

  • Multiplex staining with VASH1 antibodies and endothelial markers (CD31, CD34)

  • Quantify microvessel density, pericyte coverage, and vascular maturation

These approaches, combined with appropriate VASH1 antibody selection, enable comprehensive investigation of VASH1's complex roles in tumor angiogenesis regulation.

How can researchers effectively investigate potential contradictions in VASH1 function across different cancer types?

The contradictory roles of VASH1 across cancer types present unique research challenges requiring specialized approaches:

• Tissue-specific expression analysis:

  • Use immunohistochemistry with validated VASH1 antibodies across multiple cancer types

  • Create tissue microarrays for high-throughput comparison

  • Correlate expression patterns with clinical outcomes in each cancer type

• Isoform-specific investigation:

  • Use RT-PCR with isoform-specific primers to determine VASH1A vs. VASH1B expression ratios

  • Develop and validate isoform-specific antibodies

  • Compare functional effects of each isoform in different cancer models

• Tumor microenvironment characterization:

  • Analyze VASH1 expression in relation to immune infiltration using bioinformatic tools like CIBERSORT and ESTIMATE

  • Perform multiplex immunofluorescence to map VASH1 expression relative to various cell types

• Mechanistic studies of context-dependent effects:

  • Investigate signaling pathway interactions in different cancer types

  • Assess how VASH1 affects microtubule formation in context-specific manners

  • Examine the impact of tumor-specific mutations on VASH1 function

• Systematic meta-analysis:

  • Pool data across multiple studies with standardized antibody protocols

  • Account for methodological differences in VASH1 detection

  • Stratify results by cancer type, stage, and molecular subtype

This comprehensive approach helps reconcile seemingly contradictory findings and establish a nuanced understanding of VASH1's context-dependent roles.

What critical parameters should be evaluated when selecting a VASH1 antibody for specific research applications?

Selecting the appropriate VASH1 antibody requires careful consideration of multiple parameters:

• Target specificity:

  • Ability to distinguish VASH1 from VASH2

  • Recognition of specific VASH1 isoforms (VASH1A vs. VASH1B) if relevant

  • Epitope location and accessibility in native protein

• Host species and antibody type:

  • Polyclonal antibodies (like the Rabbit Polyclonal Antibody CAB6148) offer higher sensitivity

  • Monoclonal antibodies provide better specificity and batch-to-batch consistency

  • Host species should be chosen to minimize cross-reactivity in your experimental system

• Validated applications:

  • Confirm validation for your intended application (WB, ELISA, IHC, etc.)

  • Check recommended dilutions for each application (e.g., CAB6148: WB, 1:500-1:2000)

• Immunogen details:

  • Verify the immunogen used to generate the antibody (e.g., CAB6148 uses amino acids 1-365 of human VASH1)

  • Ensure the immunogen corresponds to conserved regions if working across species

• Sequence alignment:

  • Review the full protein sequence recognized by the antibody

  • Assess conservation of the target sequence across species of interest

• Publication record:

  • Examine literature using the antibody for similar applications

  • Evaluate performance reports in comparable experimental systems

Thorough evaluation of these parameters ensures selection of an antibody that will yield reliable, reproducible results for your specific research needs.

What are the most effective methods for validating VASH1 antibody specificity and sensitivity?

Rigorous validation of VASH1 antibodies is essential for experimental reliability:

• Positive and negative controls:

  • Use cell lines with confirmed VASH1 expression (e.g., U-251 glioma cells show high expression)

  • Include negative controls (tissue or cell line with minimal VASH1 expression)

  • Test in VASH1 knockdown/knockout systems generated via shRNA or CRISPR

• Western blot validation:

  • Confirm detection of protein at expected molecular weight (~42 kDa for VASH1)

  • Compare signal between samples with varying VASH1 expression levels

  • Evaluate ability to distinguish VASH1 isoforms if relevant

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide or recombinant VASH1

  • Observe elimination of specific signal while non-specific signals remain

• Orthogonal validation:

  • Correlate protein detection with mRNA expression (qRT-PCR)

  • Compare results from multiple antibodies targeting different VASH1 epitopes

  • Confirm with mass spectrometry if possible

• Cross-reactivity assessment:

  • Test for potential cross-reactivity with VASH2

  • Evaluate specificity across relevant species

• Dilution optimization:

  • Perform titration experiments to determine optimal concentration

  • For CAB6148, test within the recommended 1:500-1:2000 range for Western blotting

• Signal-to-noise optimization:

  • Adjust blocking conditions to minimize background

  • Optimize washing protocols to enhance specific signal

These validation steps ensure that observed signals truly represent VASH1 rather than artifacts or cross-reactive proteins.

How can researchers optimize protocols for detecting low-abundance VASH1 expression?

Detecting low-abundance VASH1 expression requires specialized approaches:

• Sample enrichment techniques:

  • Immunoprecipitation before Western blotting

  • Subcellular fractionation to concentrate VASH1-containing compartments

  • Proximity ligation assay for in situ detection of low-abundance proteins

• Signal amplification methods:

  • Tyramide signal amplification for immunohistochemistry/immunofluorescence

  • Enhanced chemiluminescence (ECL) substrates with higher sensitivity for Western blotting

  • Poly-HRP detection systems

• Optimal antibody selection:

  • Choose antibodies with higher affinity (often monoclonal)

  • Consider using cocktails of antibodies targeting different VASH1 epitopes

  • Validate sensitivity thresholds with recombinant VASH1 protein dilution series

• Protocol optimization:

  • Extended primary antibody incubation (overnight at 4°C)

  • Reduced washing stringency (shorter washes, lower salt concentration)

  • Optimize blocking conditions to reduce background while preserving signal

• Enhanced detection systems:

  • Digital immunoassays with single-molecule detection capabilities

  • Capillary Western systems with higher sensitivity than traditional Western blotting

  • Quantitative immunofluorescence with high-sensitivity cameras

• RNA-based complementary approaches:

  • RNAscope in situ hybridization to detect VASH1 mRNA

  • Single-cell RT-PCR for rare cell populations

  • Digital droplet PCR for absolute quantification

For tissues with expected low VASH1 expression (like certain normal tissues mentioned in search result ), these optimized approaches can provide reliable detection while maintaining specificity.

What are the optimal protocols for Western blot analysis of VASH1 expression?

For optimal Western blot detection of VASH1, follow this detailed protocol:

• Sample preparation:

  • Extract proteins using RIPA buffer with protease inhibitor cocktail

  • Determine protein concentration using BCA or Bradford assay

  • Prepare 20-50 μg protein per lane in Laemmli buffer with reducing agent

  • Heat samples at 95°C for 5 minutes

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal resolution of VASH1 (~42 kDa)

  • Include molecular weight markers

  • Run at 100V until samples enter resolving gel, then 150V until completion

• Transfer conditions:

  • Use PVDF membrane (0.45 μm pore size)

  • Transfer at 25V overnight at 4°C for complete transfer

  • Verify transfer efficiency with Ponceau S staining

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

  • For CAB6148, dilute 1:500-1:2000 in blocking buffer

  • Incubate with primary antibody overnight at 4°C with gentle rocking

  • Wash 4 × 5 minutes with TBST

  • Incubate with appropriate HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour

  • Wash 4 × 5 minutes with TBST

• Signal detection:

  • Apply ECL substrate according to manufacturer's instructions

  • Expose to X-ray film or use digital imaging system

  • Include both short and long exposures to capture range of expression

• Controls and normalization:

  • Include positive control (e.g., U-251 glioma cell lysate)

  • Include VASH1 knockdown control if available

  • Probe for loading control (β-actin, GAPDH) on same membrane

  • Normalize VASH1 signal to loading control for quantification

This optimized protocol provides reliable detection of VASH1 protein while minimizing non-specific background signal.

How should immunohistochemistry protocols be adapted for optimal VASH1 detection in tissue samples?

For effective VASH1 immunohistochemistry in tissue samples:

• Tissue preparation:

  • Use 4% paraformaldehyde fixation for 24 hours

  • Process and embed in paraffin according to standard protocols

  • Section at 4-5 μm thickness onto positively charged slides

  • Include known VASH1-positive tissue (e.g., tumor endothelium) as positive control

• Antigen retrieval (critical for VASH1):

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Pressure cook for 3 minutes at full pressure

  • Allow slides to cool in buffer for 20 minutes

• Blocking steps:

  • Block endogenous peroxidase with 3% hydrogen peroxide for 10 minutes

  • Block non-specific binding with 5% normal goat serum for 1 hour

  • For highly vascular tissues, consider avidin/biotin blocking

• Primary antibody:

  • Optimize dilution through titration experiments (start with manufacturer's recommendation)

  • Incubate overnight at 4°C in a humidified chamber

  • Include negative control by substituting primary antibody with isotype control

• Detection system:

  • Use polymer-based detection system for enhanced sensitivity

  • Develop with DAB substrate for 5-10 minutes (monitor microscopically)

  • Counterstain with hematoxylin (30 seconds)

  • Dehydrate through graded alcohols and clear in xylene

• Scoring and analysis:

  • Assess VASH1 staining pattern (cytoplasmic, nuclear, membranous)

  • Quantify expression using H-score (percentage × intensity)

  • Compare expression between different tissue regions and cell types

• Validation in patient samples:

  • Correlate staining with clinical parameters as done in LGG studies

  • Perform survival analysis based on VASH1 expression levels

These optimized protocols have been successfully employed to detect VASH1 in various cancer tissues, including lower-grade gliomas where VASH1 expression has prognostic significance.

What experimental design is recommended for studying VASH1's role in the tumor microenvironment?

A comprehensive experimental design for studying VASH1 in the tumor microenvironment should include:

• Spatial expression mapping:

  • Multiplex immunofluorescence combining VASH1 antibodies with:

    • Endothelial markers (CD31, CD34)

    • Immune cell markers (CD4, CD8, CD68, CD163)

    • Cancer cell markers (tumor-specific antigens)

  • Digital spatial profiling to quantify VASH1 expression across tissue regions

  • 3D reconstructions to visualize VASH1 in relation to vasculature

• Immune microenvironment analysis:

  • Apply CIBERSORT and ESTIMATE algorithms to correlate VASH1 expression with immune infiltration

  • Flow cytometry with VASH1 antibodies to identify VASH1-expressing immune populations

  • Single-cell RNA sequencing to identify cell populations expressing VASH1

• Functional studies in co-culture systems:

  • Co-culture tumor cells with endothelial cells ± VASH1 manipulation

  • Add immune components (e.g., macrophages, T cells) to assess VASH1's impact

  • Use transwell systems to distinguish direct vs. indirect effects

• Genetic manipulation approaches:

  • VASH1 knockdown in tumor and/or stromal cells

  • Isoform-specific overexpression to distinguish VASH1A vs. VASH1B effects

  • CRISPR-Cas9 editing to create VASH1 knockout models

• In vivo models with microenvironment focus:

  • Humanized mouse models with human immune components

  • Window chamber models for real-time imaging of VASH1 and microenvironment

  • Sequential sampling to track temporal changes

• Microtubule dynamics assessment:

  • Live-cell imaging with fluorescently labeled tubulin

  • Measure microtubule formation rates in presence/absence of VASH1

  • Correlate with cell migration and invasion capabilities

This comprehensive approach enables researchers to dissect the complex roles of VASH1 in shaping the tumor microenvironment, potentially identifying new therapeutic strategies.

How can researchers effectively design VASH1 knockdown experiments to study its function?

Designing effective VASH1 knockdown experiments requires careful planning:

• Selection of knockdown approach:

  • shRNA for stable long-term knockdown (as used in U-251 glioma cells)

  • siRNA for transient effects

  • CRISPR-Cas9 for complete knockout

  • Inducible systems for temporal control

• Target sequence design:

  • Design multiple shRNA/siRNA sequences targeting different VASH1 regions

  • Consider isoform-specific targeting if studying VASH1A vs. VASH1B

  • Check for potential off-target effects using bioinformatic tools

  • Include non-targeting control sequences

• Delivery optimization:

  • For glioma cells, lentiviral vectors have proven effective

  • Optimize MOI (multiplicity of infection) for maximum knockdown with minimal toxicity

  • Include GFP or other selection markers to identify transduced cells

• Validation of knockdown efficiency:

  • qRT-PCR to confirm mRNA reduction (as performed in U-251 cells)

  • Western blot with validated VASH1 antibodies to verify protein reduction

  • Aim for >80% reduction for functional studies

• Functional assessments based on known VASH1 roles:

  • Cell proliferation: MTT/WST-1 assay, cell counting, BrdU incorporation

  • Migration: Wound healing/scratch assay, transwell migration

  • Invasion: Matrigel invasion assay, 3D spheroid invasion

  • Angiogenesis: Tube formation, co-culture with endothelial cells

• Pathway analysis:

  • Assess effects on microtubule formation

  • Examine changes in immune infiltration markers

  • Evaluate angiogenesis-related signaling pathways

• Rescue experiments:

  • Re-express VASH1 (knockdown-resistant) to confirm specificity

  • Express individual VASH1 isoforms to determine their specific contributions

The study of VASH1 in U-251 glioma cells demonstrated that knockdown increased cell proliferation, invasion, and migration capacity, providing important insights into VASH1's tumor-suppressive functions in glioma .

What methodological approaches can distinguish the functions of different VASH1 isoforms?

Distinguishing VASH1 isoform functions requires specialized methodological approaches:

• Isoform-specific detection:

  • Design qRT-PCR primers spanning exon junctions unique to each isoform

  • Develop isoform-specific antibodies targeting unique regions

  • Use mass spectrometry to identify isoform-specific peptides

• Recombinant expression systems:

  • Generate expression constructs for individual VASH1 isoforms

  • Create cell lines stably expressing single isoforms

  • Use inducible systems to control expression timing and levels

• CRISPR-based approaches:

  • Design guide RNAs targeting isoform-specific exons

  • Create isoform-specific knockout models

  • Use base editing to introduce isoform-specific mutations

• Comparative functional assays:

  • Compare effects of VASH1A vs. VASH1B on:

    • Endothelial cell growth, migration, tube formation

    • Cancer cell proliferation, invasion, apoptosis

    • Immune cell function and infiltration

• Interaction studies:

  • Identify isoform-specific binding partners using co-immunoprecipitation

  • Perform yeast two-hybrid screening with individual isoforms

  • Use proximity labeling methods (BioID, APEX) to identify localized interactors

• In vivo models:

  • Generate transgenic models expressing specific isoforms

  • Use AAV vectors for tissue-specific isoform expression

  • Analyze differential effects on tumor growth and angiogenesis

Research indicates VASH1B may be more potent than VASH1A in inhibiting endothelial cell growth and inducing apoptosis in proliferating fibroblasts and cancer cells . These methodological approaches enable researchers to further delineate the distinct functions of each isoform.

How can researchers investigate the contradictory findings regarding VASH1's role in cancer progression?

Investigating contradictory findings regarding VASH1 in cancer requires systematic methodological approaches:

• Context-dependent expression analysis:

  • Compare VASH1 expression across multiple cancer types using standardized methods

  • Correlate with molecular subtypes within each cancer

  • Analyze relationship between VASH1 expression and tumor stage/grade

• Comprehensive isoform profiling:

  • Determine VASH1A:VASH1B ratios across cancer types

  • Analyze whether contradictions relate to differential isoform expression

  • Investigate cancer-specific splice variants

• Microenvironment characterization:

  • Analyze VASH1 expression in relation to:

    • Vascular density and morphology

    • Immune cell infiltration profiles

    • Hypoxic vs. normoxic regions

  • Use algorithms like CIBERSORT and ESTIMATE to quantify microenvironment components

• Mechanistic pathway analysis:

  • Compare signaling pathways activated by VASH1 across cancer types

  • Identify cancer-specific interaction partners

  • Investigate post-translational modifications affecting VASH1 function

• Integrated multi-omics approach:

  • Correlate VASH1 protein expression with transcriptomics data

  • Integrate with mutation profiles and copy number alterations

  • Analyze epigenetic regulation of VASH1 across cancer types

• Meta-analysis methodology:

  • Systematically review all VASH1 cancer studies using consistent criteria

  • Account for methodological differences in detection methods

  • Stratify by cancer type, patient demographics, and treatment history

This methodological framework helps reconcile the paradoxical findings where VASH1 acts as a tumor suppressor in some contexts (inducing apoptosis in colon cancer cells) while correlating with poor prognosis in others (LGG and breast cancer) .