Association of PP2A and VE-cadherin in a mono and co-culture with implications on brain microvascular permeability
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CASEY, GILLIAN, Association of PP2A and VE-cadherin in a mono and co-culture with implications on brain microvascular permeability, Trinity College Dublin.School of Medicine, 2019Download Item:
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Abstract:
Introduction: Based on the knowledge that VE-cadherin is an integral component of the functioning endothelium in the blood brain barrier. Its transcellular abundance can be altered by post-translational modifications and hence why it is of vital importance we understand the mechanism of VE-cadherin regulation. PP2A is the most abundant phosphatase in the brain and its loss is associated with neurological diseases such as Alzheimer’s disease. What remains undetermined are the association between PP2A and VE-cadherin in brain microvascular endothelial cells and their effect on vascular permeability.
Materials and Methods: A primary and immortal human brain microvascular endothelial cell lines were used (hBMEC and hCMEC/d3). PP2Ac activity was determined by an immunoprecipitation activity assay (Millipore). Cells were exposed to Okadaic acid (OA, 10nM) and FTY-720 (5μM). Protein abundance and mRNA expression was determined by Western-blot and RT-PCR respectively. Permeability of the endothelial monolayer was determined by monitoring the transcellular movement of a FITC labelled dextran. Proteins were overexpressed in hCMEC/d3s (CIP2A, SET and PP2A) by the transfection of the relevant overexpression plasmids for 72 h. PBMCs were isolated from whole blood and polarised into Mθ and M1 pro-inflammatory macrophages. All data were normalised to the appropriate control and expressed as an absolute value, a percentage or ratio. Data were analysed by one-way anova or two-way anova with post hoc analysis (Bonferroni).
Results: PP2A activity was inhibited by OA (10 nM), CIP2A and SET overexpression and the co-culture with M1 macrophages. Through the demethylation and phosphorylation of PP2A. Reduced PP2A activity results in the phosphorylation of VE-cadherin (Ser665) and disassociation of PP2A, α- and β- catenin from VE-cadherin. VE-cadherin undergoes clathrin mediated endocytosis, ubiquitination and proteasomal degradation. P120 and α-catenin are degraded as a result of PP2A inhibition. This results in an increase in endothelial cell paracellular permeability. Alternatively, Mθ and overexpression of PP2A results in an increase in PP2A activity through the methylation and dephosphorylation of the PP2A catalytic subunit. As PP2A activity is maintained, so is the integrity of the VE-cadherin interactome. Thus maintaining endothelial barrier integrity.
Conclusion: Overall, the results of this thesis demonstrated a strong association between PP2A activity and abundance of VE-cadherin and brain microvascular permeability. Gaining physiologically relevant understanding on how pro-inflammatory macrophages regulate PP2A, VE-cadherin and induce permeability. Unveiling how PP2A modulation alters the component of the VE-cadherin interactome has highlighted potential therapeutic targets for the prevention of brain microvascular endothelial dysfunction.
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Author: CASEY, GILLIAN
Advisor:
Spiers, JamesPublisher:
Trinity College Dublin. School of Medicine. Discipline of Pharmacology & TherapeuticsType of material:
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