AnEVs induce vascular permeability
Vascular permeability is one of the processes tightly associated to the
anaphylactic reactions [28]. To analyze a plausible role of AnEVs in
endothelial permeability, in vitro assays combined with
immunofluorescence staining were carried. Images showed that an
interaction between the PKH67-stained EVs and ECs exists. A clear
perinuclear EVs localization revealed that HMVEC-Ls engulf and
internalize these particles (Figure 5A-B ). Once confirmed that
AnEVs and HMVEC-Ls interact with each other, the purpose was to evaluate
the functional effect of these vesicles on the endothelial barrier.
Vascular permeability assays showed that the incubation of EVs extracted
from anaphylactic patients produced a decrease in the resistance of the
endothelial monolayer (Figure 5C-D ). Importantly, this analysis
including 16 patients demonstrated that AnEVs induced a greater increase
of the cellular permeability compared to the BEVs (Figure 5D ).
As control of the cellular system created, vasoactive agents were also
evaluated showing the effect of thrombin and platelet-activating factor
(PAF) as destabilizers of the monolayer and the AMPc as an endothelial
stabilizer. In all cases the expected effect was observed
(Supplemental Figure 1 ).