Endothelial dysfunction and P2X7 antagonism in TToP system
Atherosclerosis(2023)
Abstract
Background and Aims: Mechanisms promoting endothelial dysfunction include the activation of P2X7 and TNF-α pathways. Technologically advanced systems for modeling endothelial barrier represent useful tools for the study of mechanism regulating atherosclerosis development. Our aim was to investigate the relationship between P2X7 antagonism and TNF-α in endothelial dysfunction Methods: We have developed a microphysiological system called True Tissue On Platform (TToP), i.e. a bicompartmental platform with an open-well design based on laser micromachined cartridge, allowing sample retrieval without cell damage. EaHy926 endothelial cells (EC) were settled (3.5x104 cell/system) over a polycarbonate membrane allocated into the cartridge. Live imaging on Acridine Orange (AO) labeled EC assessed viability and distribution. P2X7 antagonist (A740003, 100mM for 2h) was applied in the upper chamber of TToPs to modulate the effect of TNF-a (100ng/ml, overnight). EC monolayers were destinated to immunofluorescence for confocal microscopy, or dedicated to RT-qPCR Results: AO labeling before EC treatment demonstrated the formation of tight monolayers of homogenously distributed. TNF-α significantly up-regulated IL-1β (p=0.0029) and NF-kB (p=0.0059) genes, and variably affected P2X7. Confocal microscopy in TToP cartridge with CD31/PECAM or von Willebrand Factor antibodies confirmed the phenotype preservation, that with phalloidin-TRITC showed cytoskeletal stress following TNF-α. Addition of A740003 either prior/after EC treatment with TNF-α globally reverted the activation status, significantly restoring NF-kB gene expression (p=0.003). Conclusions: Preliminary data suggest TToP suitability to study EC dysfunction and perform pharmacological approaches aimed at modulating atherosclerosis relevant pathways. In perspective, we envisage the development of more complex vessel models, including a dynamic system to study cells under perfusion/chemical gradients.
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Key words
endothelial dysfunction,p2x7 antagonism
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