ИСТИНА

Background: Paroxysmal Permeability Diseases (PPD) are conditions determined by recurrent increase of endothelial permeability with no inflammatory, degenerative, ischemic vascular injury. They can be self-limiting but also rapidly lethal (eg severe laryngeal crises of primary angioedema or hypovolemic shock in idiopathic systemic capillary leak syndrome). Aim: to investigate whether a three dimensional dynamic model, the “Microvasculature on a chip”, can be implemented to study endothelial cell barrier function, with a focus on factors which can increase/decrease the permeability of endothelial cells exposed to shear stress under physiologic flow conditions. Materials and Methods: Microchannel networks (smallest 30μm square section) were fabricated using a soft-lithography technique, as described by Tsvirkun et al. [1]. High-affinity interactions between ligand (FITC-conjugated avidin) and substrate-bound acceptor (biotin) were evaluated to assess permeability changes [2]. A polydimethylsiloxane (PDMS) upper part was sealed to a glass coverslip at the bottom. The inner surfaces of the channels were coated with biotinylated fibronectin and Human Umbilical Vein Endothelial Cells (stained with CellTracker™ Red dye) were cultured within the networks, in the presence of a steady flow of culture medium. The cells in the circuits were challenged for 15 minutes with bradykinin in culture medium or human plasma (from a healthy volunteer) +FITC-avidin solution. Afterwards, circuits were fixed in 4% paraformaldehyde. The control circuit was exposed only to culture medium. Confocal Fluorescence Microscopy was used for cells’ imaging. The acquired image stacks with 1µm step were processed and analyzed using the Fiji open-source platform. Results: The circuit challenged with bradykinin showed more average/peak fluorescence intensity (which is proportional to endothelial permeability) as compared to the circuit challenged with plasma and to the control. Conclusion: The “Microvasculature on a chip” model allows to assess alterations of endothelial permeability under dynamic conditions and it opens new perspectives for investigating the pathophysiology of PPD.