Abstract Introduction Vascular complications in diabetes are characterized by endothelial dysfunction and leukocyte anomalies driven by intricate cellular events associated with elevated blood glucose levels. Purpose This project explores the function of extracellular vesicles (EVs) in intercellular communication between endothelial cells (ECs) and macrophages (Mø) in a diabetic environment. Methods EVs were extracted from HUVEC and patient-derived HCMEC cultivated in normal glucose (NG) or high glucose (HG) media. Nanoparticle tracking analysis (NTA) was conducted to assess EV quality, and THP1-derived macrophages were stimulated with NG-EC-EVs or HG-EC-EVs. Uptake assays, functional characterization, and RNA-Seq analysis were performed on EVs exposed THP1 cells. Proteomic analysis was carried out to elucidate the EV content profile. Results THP1-derived macrophages exposed to HG-EC-EVs displayed a pronounced pro-inflammatory phenotype, evidenced by elevated M1 markers (TNF-α, iNOS) and reduced M2 marker (MRC) expression. RNA-Seq analysis revealed substantial alterations in gene expression patterns, with upregulation of pro-inflammatory pathways including cytokine-cytokine interaction, TNF signaling, and JAK-STAT signaling. Crucial genes such as TNFSF15 and MAPK10 were significantly upregulated. Utilizing the MacID classification model, we demonstrated a distinct LPS, IL10 signature. The transcriptional modifications were accompanied by functional changes, with HG-EC-EVs triggering NF-κB activation. Functional assays corroborated pro-inflammatory effects, demonstrating increased TNF-α and IL-1β secretion. Fluorescent EV uptake assays showed similar EV internalization between NG and HG conditions, suggesting that the observed effects were attributable to EV content rather than uptake efficiency. EV content analysis via proteomics unveiled upregulation of pathways related to cardiac function, extracellular matrix interactions, and AGE-RAGE signaling. Conclusion Our results demonstrate that hyperglycemic conditions significantly alter EC-EV profiles, promoting macrophage polarization towards a pro-inflammatory phenotype. These findings underscore the pivotal role of EVs in modulating inflammatory responses in diabetic conditions and offer potential targets for therapeutic intervention.
Germena et al. (Sat,) studied this question.