PECAM1 Mouse

What is the structure and expression pattern of PECAM1 in mice?

PECAM1 in mice is a transmembrane glycoprotein with extracellular, transmembrane, and cytoplasmic domains. The mouse PECAM1 protein spans from Glu18 to Lys590, with homology to human PECAM1 . It is predominantly expressed on:

• Vascular endothelial cells (particularly concentrated at intercellular junctions)

• Platelets

• Subsets of leukocytes, including neutrophils, monocytes, and NK cells

• Some lymphocyte subpopulations

Expression levels vary by tissue type, with highest expression in highly vascularized tissues. The protein functions as a cell adhesion molecule and participates in cellular signaling pathways critical for vascular integrity and immune cell migration .

How are PECAM1 knockout mice generated and validated?

PECAM1 knockout mice are generated through several approaches:

Global Knockout Method:

• Target the PECAM1 gene (particularly exons 1 and 2) through homologous recombination in embryonic stem cells

• Select positive ES cell clones using neomycin resistance

• Inject targeted ES cells into C57BL/6 blastocysts

• Implant blastocysts into pseudo-pregnant females

• Breed chimeric offspring to establish germline transmission

• Confirm knockout through PCR genotyping, Western blotting, and immunohistochemistry

Conditional Knockout Method:

• Generate a targeting construct containing loxP sites flanking PECAM1 exons 1 and 2

• Include a neomycin resistance gene flanked by FRT sites

• Electroporate into C57BL/6 embryonic stem cells

• Select and validate correctly targeted ES cell clones

• Generate chimeric animals by blastocyst injection

• Breed with FLP1-expressing mice to remove the neomycin cassette

• Cross PECAM1^flox/flox mice with tissue-specific Cre recombinase-expressing mice

Validation typically involves confirming loss of PECAM1 expression through immunostaining, flow cytometry, and Western blot analysis .

What phenotypic characteristics are observed in PECAM1 knockout mice?

PECAM1 knockout mice exhibit several notable phenotypic characteristics:

These mice develop normally but show significant alterations in inflammatory responses and vascular integrity maintenance, indicating PECAM1's critical role in these processes .

How does PECAM1 deficiency affect vascular permeability in mouse models?

PECAM1 deficiency significantly impacts vascular permeability regulation:

Mechanistic Basis:

• PECAM1-deficient endothelial cells exhibit prolonged permeability changes in response to histamine treatment compared to PECAM1-reconstituted cells

• PECAM1 knockout mice demonstrate exaggerated and prolonged CNS vascular permeability during the development of EAE

• There is a delay in restoration of dermal vascular integrity following histamine challenge in PECAM1 knockout mice

Molecular Mechanisms:

• PECAM1 stabilizes endothelial junctions through homophilic binding

• It participates in signaling pathways that restore barrier function after inflammatory challenges

• The protein interacts with cytoskeletal components to maintain junction integrity

• PECAM1 modulates VE-cadherin-based adherens junction stability

Experimental Approach for Studying Permeability:

• Evans blue extravasation assay to quantify vascular leak

• Intravital microscopy to visualize real-time permeability changes

• Transwell permeability assays with isolated endothelial cells

• Measurement of transendothelial electrical resistance (TEER)

The impaired barrier restoration in PECAM1-deficient models underscores its role as a negative regulator of vascular permeability and importance in maintaining vascular homeostasis .

What is the role of PECAM1 in experimental autoimmune encephalomyelitis (EAE) mouse models?

PECAM1 plays a crucial regulatory role in EAE progression:

Key Findings:

• PECAM1-deficient mice exhibit an earlier onset of clinical symptoms during EAE compared to wild-type counterparts

• During EAE, mononuclear cell extravasation and CNS infiltration occur at earlier time points in PECAM1-KO mice

• The absence of PECAM1 leads to enhanced T lymphocyte transendothelial migration in vitro

Mechanistic Contributions:

• PECAM1 regulates the integrity of the blood-brain barrier (BBB) during inflammatory challenges

• It modulates the kinetics and extent of immune cell infiltration into the CNS

• PECAM1 influences the restoration of vascular integrity after inflammatory insult

Experimental Approaches:

• MOG immunization model for chronic EAE induction in C57BL/6 mice

• Clinical scoring to assess disease progression

• Histopathological examination of CNS tissues for immune cell infiltration

• In vitro transendothelial migration assays with T cells across PECAM1-deficient endothelial monolayers

• Assessment of BBB permeability using tracer molecules

The EAE model in PECAM1-deficient mice reveals its importance in regulating both immune cell trafficking and vascular integrity in the context of neuroinflammation .

How does PECAM1 interact with PIEZO1 at cellular junctions in mouse models?

Recent research has uncovered a significant interaction between PECAM1 and PIEZO1:

Interaction Characteristics:

• PIEZO1 and PECAM1 physically interact at cell-cell junctions, as demonstrated by FRET analysis

• This interaction is mediated by specific C-terminal residues in PECAM1, particularly Y713

• The Y713F mutation in PECAM1 prevents FRET signaling with PIEZO1 and reduces PIEZO1 recruitment to junctions

Functional Consequences:

• PECAM1 inhibits PIEZO1 mechanical sensitivity, shifting the pressure-response curve to the right

• PECAM1 reduces PIEZO1 activity measured as total charge flow per pressure step

• This interaction potentially serves as a mechanical sensitivity regulator in endothelial junctions

Experimental Approaches:

• Patch-clamp recordings of PIEZO1 channel activity in the presence/absence of PECAM1

• FRET analysis using fluorescently tagged PIEZO1 and PECAM1

• Site-directed mutagenesis of key residues (Y713F, C622A, S700F)

• Pressure-response curve analysis to quantify mechanical sensitivity

• Localization studies with tagged proteins to identify junctional enrichment

This interaction represents a novel mechanism by which PECAM1 regulates mechanosensitivity at vascular junctions, with implications for endothelial responses to flow and pressure .

What are the approaches for creating and utilizing conditional PECAM1 knockout mice?

Conditional PECAM1 knockout mice provide sophisticated tools for tissue-specific analysis:

Generation Strategy:

• Creation of a targeting construct with loxP sites flanking PECAM1 exons 1 and 2

• Incorporation of a neomycin resistance gene flanked by FRT sites positioned upstream of the 3' loxP site

• Electroporation into C57BL/6 embryonic stem cells

• Injection of correctly targeted ES cells into blastocysts

• Breeding chimeric animals with FLP1-expressing mice to remove the neomycin cassette

• Generation of homozygous PECAM1^flox/flox mice

Conditional Knockout Applications:

• Crossing with Sox2Cre mice to generate global knockouts

• Crossing with Tie2Cre for endothelial/hematopoietic-specific deletion

• Crossing with CD11cCre for dendritic cell-specific deletion

• Crossing with LysMCre for myeloid cell-specific deletion

Validation and Characterization:

• PCR genotyping to confirm presence of floxed alleles and Cre recombinase

• Western blot analysis to verify tissue-specific PECAM1 deletion

• Immunofluorescence to visualize cellular-specific deletion patterns

• Functional assays to assess tissue-specific consequences

This approach enables researchers to distinguish the roles of PECAM1 in different cell types and overcome the limitations of global knockout models where compensatory mechanisms may mask phenotypes .

How does PECAM1 influence leukocyte transmigration in different inflammatory scenarios?

PECAM1's role in leukocyte transmigration is highly context-dependent:

Leukocyte Subtype Specificity:

• PECAM1 mediates transmigration of monocytes, neutrophils, and NK cells

• Only specific subsets of lymphocytes utilize PECAM1 for transmigration

Stimulus Specificity:

Mechanistic Contributions:

• PECAM1 facilitates migration through the endothelial junction via homophilic interactions

• It mediates migration through the basement membrane via PECAM1-integrin interactions

• Involvement in intracellular signaling that regulates directional migration

Experimental Approaches:

• Intravital microscopy to visualize leukocyte-endothelial interactions

• Transwell migration assays with different inflammatory stimuli

• Parallel plate flow chamber assays to assess transmigration under flow

• Tissue-specific analysis of leukocyte infiltration in inflammatory models

This differential involvement highlights PECAM1 as a context-specific regulator of leukocyte transmigration, dependent on both the leukocyte subtype and the nature of the inflammatory stimulus .

What approaches can be used to study recombinant mouse PECAM1 protein in experimental systems?

Recombinant mouse PECAM1 proteins provide valuable tools for mechanistic studies:

Available Forms:

• Full-length extracellular domain (Glu18-Lys590)

• Domain-specific constructs

• Tagged versions (e.g., with C-terminal 6-His tag)

Experimental Applications:

• Binding Studies:

  • Solid-phase binding assays to identify interaction partners

  • Surface plasmon resonance to determine binding kinetics

  • Co-immunoprecipitation experiments with potential binding partners

• Functional Assays:

  • Blocking antibody studies to inhibit specific interactions

  • Competition assays with soluble PECAM1 versus membrane-bound forms

  • Reconstitution experiments in PECAM1-deficient systems

• Structural Studies:

  • Crystallography of PECAM1 domains

  • Cryo-EM for larger complexes

  • Domain mapping for interaction surfaces

• In vivo Administration:

  • Competitive inhibition of PECAM1-dependent processes

  • Tracking studies with labeled recombinant protein

  • Rescue experiments in PECAM1-deficient animals

Quality Control Parameters:

• Protein activity measured by binding assays (ED₅₀ of 0.060-0.720 μg/mL)

• Purity assessment by SDS-PAGE

• Endotoxin testing for in vivo applications

Recombinant PECAM1 proteins serve as essential tools for dissecting molecular mechanisms in both cell culture and in vivo mouse models .