Figure 1.

Schematic overview of study workflow

(A) Primary brain endothelial cells (ECs) were cultured under static conditions without TNFα (HBMEC^static) or with TNFα (HBMEC^static+TNF) as were primary lung ECs (HMVEC-L^static or HMVEC-L^static+TNF). Culture supernatants containing EVs were harvested and sequentially centrifuged to isolate EVs.

(B) Transmission electron microscopy was performed to confirm the presence of EVs after purification.

(C) A laminar flow system was used to apply shear stress of 1.5 dyne/cm². Stimuli were then added to the fluidic unit: (i) incubation at 40°C (ECs^1.5+40°C), (ii) non-infected red blood cells (RBCs) (ECs^1.5+RBCs), and (iii) infected RBCs (iRBCs) (ECs^1.5+Rings).

(D) mRNA/miRNAs were then isolated from ECs and purified EVs and subjected to NGS sequencing.

(E) Raw data were extracted and subjected to RNA-seq analysis and miRNA counting using a CLC genomics workbench. Subsequently, differential expression analysis was performed. Finally, miRNA target filtration was performed followed by pathway analysis using Reactome and KEGG pathway analysis.