ANXA5 Mouse

What is ANXA5 and what are its key structural features in mice?

ANXA5 (Annexin A5) is a single-chain phospholipid-binding protein belonging to the annexin gene superfamily. In mice, it is encoded by the Anxa5 gene with accession number P48036 . The recombinant mouse ANXA5 protein has a calculated molecular mass of approximately 36 kDa, with its expressed region typically spanning from Ala2 to Asp319 . ANXA5 is characterized by its high-affinity binding to phosphatidylserine (PS) in a calcium-dependent manner, which is central to its biological functions . The protein is also known by several synonyms including Annexin V, Anchorin CII, Calphobindin I (CBP-I), Lipocortin V, and Vascular anticoagulant-alpha (VAC-alpha) .

How does ANXA5 function differ between developmental stages in mouse models?

Research indicates significant changes in ANXA5 abundance during mouse development, particularly in specialized tissues. In vestibular hair bundles of the inner ear, ANXA5 concentration increases substantially from developmental stages to adulthood. Quantitative mass spectrometry has shown that in postnatal days 4-6 (P5) mouse utricle hair bundles, there are approximately 3,500 ANXA5 molecules per stereocilium, whereas by P21-P25 ("P23"; young adult), this increases dramatically to approximately 15,000 molecules per stereocilium . This developmental increase suggests stage-specific functions that may relate to membrane stabilization or repair mechanisms as the sensory structures mature. Unlike many developmental changes, this increase is not compensated by other annexin family members, as their combined abundance remains at approximately 1,000 molecules per stereocilium regardless of developmental stage .

How does mouse ANXA5 compare to human ANXA5 in terms of structure and function?

While the search results don't directly compare mouse and human ANXA5, research protocols reveal important insights. Studies often use human ANXA5 cDNA expressed in bacterial systems like Escherichia coli M15 for experimental applications in mouse models . This cross-species compatibility suggests significant structural and functional conservation between mouse and human ANXA5. Both function similarly in phosphatidylserine binding and show parallel anti-inflammatory properties when administered in mouse disease models . The effectiveness of human ANXA5 in mouse experimental systems indicates that the core functional domains and mechanisms are highly conserved between species, making mouse models valuable for translational research investigating ANXA5's therapeutic potential in human diseases.

What are the recommended expression and purification methods for producing recombinant mouse ANXA5?

Recombinant mouse ANXA5 can be produced through several expression systems, with mammalian and bacterial systems being the most common. For studies requiring post-translational modifications, HEK293 cell expression systems are often preferred. The protocol involves:

• Transfecting HEK293 cells with an expression vector harboring the mouse ANXA5 gene

• Growing cells in serum-free medium

• Harvesting cell culture by centrifugation to remove cells

• Collecting the supernatant containing secreted ANXA5

• Adding mammalian cell protease inhibitor cocktail

• Immediate aliquoting and storage at -80°C
  For higher yield and less expensive production, bacterial expression systems can be used:

• Transform Escherichia coli M15 (pREP4) with pQE30Xa containing mouse ANXA5 cDNA

• Harvest bacteria and lyse by sonification

• Remove cell debris by centrifugation

• Isolate His-tagged ANXA5 from supernatant using nickel columns and an imidazole gradient

• Verify protein homogeneity using MALDI-TOF/TOF

• Confirm PS binding activity through ellipsometry

• Ensure endotoxin levels are below one endotoxin unit per ml using spectrophotometry
  Both methods produce functional ANXA5, though the choice depends on experimental requirements for post-translational modifications and application.

What quantitative methods are most effective for measuring ANXA5 expression levels in mouse tissues?

Quantitative mass spectrometry has proven particularly effective for precise measurement of ANXA5 expression levels in mouse tissues. The recommended methodology includes:

• Tissue isolation (e.g., bundle isolation from mouse utricles using the twist-off technique)

• Sample preparation for shotgun mass spectrometry

• Analysis using an Orbitrap mass spectrometer

• Quantification with MaxQuant software and the iBAQ algorithm

• Measurement of peptide extracted ion chromatogram areas

• Calculation of relative molar abundance in specific tissues or cellular compartments
  This approach allows researchers to determine that ANXA5 accounts for approximately 2% of total protein in mouse vestibular hair bundles, representing about 15,000 copies per stereocilium . For relative quantification across multiple annexin family members, this method enables comprehensive profiling that reveals ANXA5 as the most abundant membrane-associated protein in certain structures, far exceeding other annexin isoforms .

How can researchers effectively generate and validate Anxa5 knockout mouse models?

While the search results don't provide a comprehensive protocol for generating Anxa5 knockout mice, they do reference the use of Anxa5^-/- mice in research, suggesting established methodologies exist . Based on standard practices and the referenced studies, an effective approach would include:

• Generation phase:

  • Use gene targeting techniques to disrupt the Anxa5 gene

  • Confirm targeting through Southern blotting and PCR genotyping

  • Establish breeding colonies with appropriate background strains

• Validation phase:

  • Confirm absence of ANXA5 protein using Western blotting

  • Verify no compensation by other annexin family members through mass spectrometry analysis

  • Assess basic health parameters including weight development, mortality rates, and blood parameters

• Functional validation:

  • Test physiological functions related to known ANXA5 roles (hearing, balance, cardiovascular function)

  • Challenge mice with appropriate stressors (e.g., noise exposure for hearing studies)
    The search results indicate that properly validated Anxa5^-/- mice show normal development, no mortality differences, and similar weight progression compared to wild-type mice, suggesting effective compensation mechanisms operate in the absence of this protein .

What evidence supports ANXA5's role in atherosclerosis progression in mouse models?

Research using ApoE^-/- mouse models provides substantial evidence for ANXA5's protective role in atherosclerosis. Administration of ANXA5 during early-onset atherosclerosis significantly reduces plaque formation and promotes plaque stability. Key experimental findings include:

• Reduced atherosclerotic development in the aortic root (130,027 ± 16,988 vs. 83,766 ± 9,408 μm², p = 0.0242) and aortic arch (221,811 ± 39,118 vs. 99,770 ± 9,922 μm², p = 0.0077) compared to control animals

• Decreased plaque formation consistently across all vessels in the aortic arch (excluding the carotid artery), suggesting a generalized mechanism rather than a flow-associated effect

• Reduced macrophage infiltration and increased expression of plaque stability markers in animals receiving ANXA5 treatment

• Diminished rate of apoptosis within plaques during early disease stages, though this effect appears to be overwhelmed in later stages of atherosclerosis
  These findings suggest a dual protective mechanism: early-stage reduction in apoptotic rates and macrophage infiltration, combined with inhibition of monocyte adhesion to the endothelial layer, collectively reducing plaque growth and promoting stability .

What is the optimal dosing regimen and administration route for ANXA5 in mouse atherosclerosis models?

Based on experimental protocols with demonstrated efficacy, the optimal dosing regimen for ANXA5 in mouse atherosclerosis models consists of:

• Dose: 1 mg/kg body weight

• Administration route: Intraperitoneal (i.p.) injection

• Frequency: Three times weekly

• Treatment initiation: After one week of Western-type diet (0.25% cholesterol)

• Treatment duration: Six weeks has shown significant effects on plaque development
  This regimen was established based on blood clearance studies determining ANXA5's half-life following i.p. injection to be 5.64 ± 2.33 hours . The dosage and administration schedule are designed to maintain therapeutic levels while accounting for clearance rates. This protocol has proven effective in reducing plaque formation in the ApoE^-/- mouse model without causing adverse effects on weight development, mortality, cholesterol levels, or circulating leukocyte subsets .

How does ANXA5 affect noise-induced hearing damage in mouse models?

Contrary to what might be expected given ANXA5's abundance in inner ear structures, research has shown that ANXA5 does not play a significant role in protecting against noise-induced hearing damage in mouse models. Despite ANXA5 being the most abundant membrane-associated protein in vestibular hair bundles (~15,000 copies per stereocilium), mature Anxa5^-/- mice show:

• Normal hearing and balance function under standard conditions

• Identical responses to noise exposure compared to wild-type mice

• No differences in temporary or permanent changes in hearing sensitivity following acoustic trauma
  These findings are particularly surprising given annexins' proposed role in calcium-dependent repair of membrane lesions, which could theoretically contribute to recovery mechanisms after noise damage. The research suggests that despite its unusual abundance, ANXA5 does not participate in the hair bundle's mechanotransduction function or in protective responses to acoustic trauma, at least until after two months of age in the cochlea and six months in the vestibular system .

How can researchers address the paradox between ANXA5 abundance and its apparent lack of functional significance in certain tissues?

The high abundance yet limited functional significance of ANXA5 in certain tissues presents an intriguing research paradox. To address this scientifically, researchers should consider the following methodological approaches:

• Age-dependent functional analyses:

  • Extend functional studies beyond the currently investigated age points (up to two months for cochlea and six months for vestibular system)

  • Implement aging studies to determine if ANXA5's role emerges in senescence-related processes

• Stress-specific challenges:

  • Test beyond acoustic trauma by examining responses to other stressors (oxidative stress, mechanical trauma, ototoxic compounds)

  • Implement multiple stress paradigms with varying intensities and durations

• Subtler phenotypic analyses:

  • Develop more sensitive assays for vestibular and auditory function beyond standard tests

  • Implement single-cell electrophysiology to detect subtle changes in mechanotransduction

  • Use high-resolution imaging to identify subcellular structural alterations

• Redundancy and compensation mechanisms:

  • Systematically inhibit multiple annexin family members simultaneously

  • Create conditional knockouts with temporal control to bypass developmental compensation

  • Perform comprehensive transcriptomic and proteomic analyses to identify upregulated compensatory pathways in ANXA5's absence
    This scientific paradox reinforces the critical concept that protein abundance does not necessarily correlate with functional significance within specific cellular structures or compartments .

What are the methodological considerations for investigating ANXA5's calcium-dependent membrane binding in live mouse tissues?

Investigating ANXA5's calcium-dependent membrane binding in live mouse tissues requires sophisticated methodological approaches that balance physiological relevance with technical feasibility:

• Calcium concentration control:

  • Implement precise calcium buffering systems calibrated to physiological ranges

  • Use calcium ionophores to experimentally manipulate calcium levels in a controlled manner

  • Employ genetically-encoded calcium indicators for spatiotemporal analysis of calcium fluctuations

• Membrane dynamics visualization:

  • Use fluorescently-tagged ANXA5 constructs (ensuring tag doesn't interfere with binding)

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to measure binding kinetics

  • Apply super-resolution microscopy techniques to visualize nanoscale membrane interactions

• Phosphatidylserine accessibility monitoring:

  • Use complementary PS-binding probes with different binding properties

  • Implement systems to induce controlled PS exposure (e.g., apoptosis inducers, membrane scrape techniques)

  • Consider dual-labeling approaches to simultaneously track PS exposure and ANXA5 binding

• Tissue-specific considerations:

  • For hair cells, develop methods to maintain viability of isolated stereocilia or hair bundles

  • For vascular tissues, consider ex vivo flow systems that maintain physiological pressure and flow

  • Develop clearing techniques compatible with calcium-dependent binding studies for whole-tissue imaging

• Quantification approaches:

  • Implement ratiometric imaging for quantitative binding analysis

  • Develop computational models to estimate binding parameters from imaging data

  • Use advanced image analysis algorithms to detect subtle changes in membrane association patterns
    These methodological considerations address the technical challenges of studying dynamic calcium-dependent processes while maintaining physiological relevance.

What are the recommended controls for ensuring specificity in ANXA5 binding studies using mouse models?

To ensure specificity in ANXA5 binding studies using mouse models, researchers should implement a comprehensive set of controls addressing multiple aspects of experimental design:

• Genetic controls:

  • Include Anxa5^-/- mice as negative controls

  • Use wild-type littermates as positive controls

  • Consider heterozygous mice to evaluate dose-dependent effects

• Protein-specific controls:

  • Use the M1234 variant (calcium-binding deficient ANXA5) as a functional negative control

  • Include other annexin family members to assess binding specificity

  • Employ competition assays with unlabeled ANXA5 to confirm binding site specificity

• Calcium-dependency controls:

  • Perform parallel experiments with calcium chelators (EGTA)

  • Include calcium titration series to establish dose-response relationships

  • Implement calcium-free conditions as negative controls

• Phospholipid specificity controls:

  • Use liposomes with defined phospholipid compositions

  • Include membranes lacking phosphatidylserine as negative controls

  • Implement competition with other PS-binding proteins (e.g., lactadherin)

• Technical controls:

  • For labeled ANXA5, include the fluorophore/tag alone to control for non-specific interactions

  • Perform pre-adsorption controls to verify antibody specificity

  • Include isotype controls for immunological detection methods

• Sample preparation controls:

  • Process tissues from different mouse strains identically

  • Implement vehicle-only controls for any treatments

  • Include time-matched controls to account for potential temporal variations
    These controls collectively ensure that observed effects are specifically attributable to ANXA5's physiological binding properties rather than experimental artifacts or non-specific interactions.

How should researchers interpret contradictory findings between in vitro and in vivo ANXA5 studies in mouse models?

When faced with contradictory findings between in vitro and in vivo ANXA5 studies in mouse models, researchers should employ a systematic analytical approach:

• Contextual analysis:

  • Examine physiological relevance of in vitro conditions (calcium concentrations, PS accessibility)

  • Consider developmental timing differences between systems

  • Evaluate the complexity of the microenvironment present in vivo but absent in vitro

• Methodological reconciliation:

  • Compare protein concentrations between systems (local concentrations in vivo may differ significantly)

  • Assess differences in post-translational modifications between systems

  • Evaluate temporal aspects (acute vs. chronic effects)

• Integrated analysis framework:

  • Consider in vitro findings as mechanistic insights rather than functional predictions

  • View in vivo results as net effects that may mask opposing mechanisms

  • Develop intermediate complexity models (ex vivo systems, organoids) to bridge the gap

• Specific examples from ANXA5 research:

  • In vitro studies show ANXA5 binding to PS-expressing cells suppresses pro-inflammatory actions

  • Flow chamber experiments demonstrate AnxA5 inhibits monocyte adhesion by 20%

  • Yet Anxa5^-/- mice show normal development and function in certain systems
    This suggests compensatory mechanisms operate in vivo that are absent in vitro, or that the threshold for functional significance differs between systems.

• Translational implications:

  • Consider pharmacological effects (administered ANXA5) versus genetic models (knockout)

  • Evaluate dose-responses that may explain different outcomes

  • Assess bioavailability and tissue distribution factors present in vivo but not in vitro
    This analytical framework helps researchers synthesize seemingly contradictory findings into a more comprehensive understanding of ANXA5 biology.

What are common technical challenges in quantifying ANXA5 expression across different mouse tissues and how can they be overcome?

Quantifying ANXA5 expression across different mouse tissues presents several technical challenges that can be addressed through methodological refinements:

• Mass spectrometry-based quantification using the iBAQ algorithm for absolute quantification

• Western blotting with carefully validated antibodies for relative expression

• Parallel mRNA quantification to assess transcriptional versus post-transcriptional regulation

• Preparation of reference standards from recombinant mouse ANXA5 for calibration curves

• Spatial mapping using immunohistochemistry to complement quantitative data
  These approaches collectively address the technical challenges in obtaining reliable quantitative data across diverse tissue contexts.

What statistical approaches are most appropriate for analyzing ANXA5 effects in atherosclerosis progression studies using mouse models?

When analyzing ANXA5 effects in atherosclerosis progression studies using mouse models, researchers should implement statistical approaches that address the specific characteristics of these experiments:

• Power analysis considerations:

  • Based on published data, atherosclerosis progression studies show substantial variability

  • Sample sizes of 16-18 animals per group have successfully detected significant differences in plaque size

  • A priori power calculations should account for expected effect sizes (30-50% reduction in plaque area)

• Primary outcome measures:

  • For plaque size: unpaired t-tests or Mann-Whitney tests depending on normality

  • For multiple vessel comparisons: ANOVA with appropriate post-hoc tests, with correction for multiple comparisons

  • For categorical data: Chi-square or Fisher's exact test

• Longitudinal data analysis:

  • Mixed-effects models to account for repeated measures

  • Analysis of rate of progression rather than endpoint measurements

  • Time-to-event analysis for development of advanced plaques

• Addressing covariates:

  • ANCOVA to control for variables like weight, cholesterol levels, and baseline measurements

  • Multivariate regression to identify predictors of treatment response

  • Stratified analysis when appropriate (e.g., by sex or baseline plaque burden)

• Dose-response relationships:

  • Implement dose-response modeling (linear, sigmoidal) when multiple doses are tested

  • Calculate EC50 values for therapeutic applications

  • Test for linearity of response

• Reporting requirements:

  • Always include exact p-values rather than thresholds

  • Report confidence intervals for primary outcome measures

  • Present individual data points alongside means and standard deviations

  • Clearly state the statistical tests used for each analysis
    These statistical approaches provide robust analysis of ANXA5 effects while accounting for the biological variability inherent in atherosclerosis studies.

What are promising research directions for understanding ANXA5's functional redundancy with other annexin family members in mouse models?

Given the lack of phenotype in Anxa5^-/- mice and evidence of limited compensation by other annexins, several promising research directions emerge:

• Systematic combinatorial knockout studies:

  • Generate double or triple knockouts of annexin family members

  • Prioritize combinations based on co-expression patterns

  • Implement conditional knockout systems to bypass embryonic lethality

• Stress-response profiling:

  • Subject Anxa5^-/- mice to various stressors (oxidative, mechanical, inflammatory)

  • Perform comprehensive transcriptomic and proteomic analyses under stress conditions

  • Identify stress-specific compensatory mechanisms

• Functional domain analysis:

  • Create knock-in mice expressing chimeric annexins (domain swapping)

  • Test whether specific domains rather than whole proteins provide functional redundancy

  • Identify critical residues for shared functions through point mutations

• Temporal dynamics investigations:

  • Implement time-resolved proteomics following annexin knockout

  • Study developmental switches in annexin expression and function

  • Use inducible knockout systems to distinguish developmental from acute compensation

• Cell-type specific analyses:

  • Perform single-cell transcriptomics to identify cell populations with unique annexin expression patterns

  • Create cell-type specific knockouts to bypass systemic compensation

  • Investigate annexin redundancy in specialized cell types (e.g., hair cells, macrophages)

• Evolution-guided approaches:

  • Conduct comparative studies across species with varying annexin repertoires

  • Identify evolutionary patterns suggesting functional redundancy

  • Test whether annexin functions are conserved across distant species
    These research directions would significantly advance our understanding of functional redundancy within the annexin family, potentially revealing context-specific roles that aren't apparent in standard knockout models.

How might advanced imaging techniques advance our understanding of ANXA5 dynamics in living mouse tissues?

Advanced imaging techniques offer transformative potential for understanding ANXA5 dynamics in living mouse tissues:

• Intravital microscopy applications:

  • Implement cranial windows for real-time imaging of ANXA5 in cerebrovascular disease models

  • Develop abdominal imaging windows for visualizing ANXA5 in atherosclerosis progression

  • Use genetically encoded calcium indicators alongside fluorescently tagged ANXA5 to correlate calcium dynamics with binding events

• Super-resolution approaches:

  • Apply STED microscopy to resolve ANXA5 clustering at nanoscale resolution

  • Implement PALM/STORM imaging to track single-molecule dynamics at the membrane

  • Use expansion microscopy to map ANXA5 distribution within complex tissue architectures

• Functional correlation techniques:

  • Combine electrophysiology with fluorescence imaging in hair cells

  • Implement tension sensors to correlate mechanical forces with ANXA5 recruitment

  • Use optogenetic calcium manipulation to trigger and observe ANXA5 translocation events

• Multi-parameter imaging strategies:

  • Implement multiplexed imaging to simultaneously track multiple annexin family members

  • Combine phosphatidylserine probes with ANXA5 imaging to correlate lipid dynamics

  • Integrate membrane tension sensors with ANXA5 imaging to test mechanosensitive binding

• Temporal resolution enhancements:

  • Apply fluorescence lifetime imaging to distinguish bound from unbound ANXA5

  • Use high-speed imaging to capture rapid membrane repair events

  • Implement light-sheet microscopy for extended time-lapse imaging with minimal phototoxicity

• Whole-animal imaging approaches:

  • Develop near-infrared ANXA5 probes for non-invasive imaging of inflammation

  • Apply PET imaging with radiolabeled ANXA5 to track distribution in disease models

  • Implement photoacoustic imaging for deeper tissue visualization of ANXA5 dynamics
    These advanced imaging approaches would provide unprecedented insights into the dynamic behavior of ANXA5 in living tissues, potentially revealing functional roles that have been missed by endpoint analyses.

What emerging therapeutic applications might exploit ANXA5's properties in mouse models of human disease?

Based on current understanding of ANXA5's properties, several emerging therapeutic applications in mouse models of human disease show promise:

• Atherosclerosis and cardiovascular applications:

  • Develop extended-half-life ANXA5 variants for less frequent dosing

  • Create targeted delivery systems for vascular-specific ANXA5 administration

  • Test ANXA5 as an adjunct therapy with statins or other standard treatments

• Inflammatory disease modulation:

  • Investigate ANXA5 for reducing inflammation in autoimmune disease models

  • Test PS-binding properties for neutralizing inflammatory extracellular vesicles

  • Develop combination approaches targeting multiple danger-associated molecular patterns (DAMPs)

• Membrane repair applications:

  • Engineer ANXA5 variants with enhanced membrane binding properties

  • Test preventive administration before exposure to membrane-damaging agents

  • Investigate tissue-specific delivery for organs vulnerable to membrane damage

• Diagnostic imaging platforms:

  • Develop ANXA5-based molecular imaging agents for detecting apoptosis in vivo

  • Create multimodal probes combining ANXA5 with various imaging reporters

  • Test ANXA5 imaging for early detection of treatment response in cancer models

• Extracellular vesicle engineering:

  • Use ANXA5 coating to reduce immunogenicity of therapeutic vesicles

  • Develop ANXA5-based purification systems for isolating specific vesicle populations

  • Create ANXA5 fusion proteins for targeting therapeutic vesicles to specific tissues

• Biomaterial applications:

  • Incorporate ANXA5 into biomaterials to improve biocompatibility

  • Develop ANXA5-coated implants for reduced inflammatory response

  • Create smart materials with calcium-dependent ANXA5 presentation These therapeutic directions build upon ANXA5's unique properties while addressing limitations in current approaches, potentially leading to novel interventions for human diseases that have been validated in mouse models.