Trimethylamine-N-Oxide (TMAO)-induced Endothelial Cells derived Exosomes contributes to Endothelial Dysfunction

Objective: Gut microbe metabolite, Trimethylamine-N-Oxide (TMAO), has been identified as an independent risk factor for adverse cardiovascular events. However, the molecular mechanisms of TMAO-induced cardiovascular diseases (CVDs) is not clearly understood. In recent years extracellular vesicles has gained attention because of their role in physiological and pathological processes in CVDs and their potential as a biomarker in prognosis of CVDs. It is unclear whether TMAO trigger the formation and release of exosomes from endothelial cells (ECs). Based on our preliminary results, we hypothesized that TMAO stimulation results in enhanced exosomal release from ECs and these EC-derived exosomes have the potential to induce endothelial dysfunction. Methods: Exosomes were isolated from the EC culture and plasma with or without the treatment of TMAO, using exosome isolation kit. Exosomes markers like CD63, CD9 were analysed by western blot. Isolated exosomes were characterized by Nano Sight NS300 analyser. Exosome’s structure and morphology were determined by transmission electron microscope (TEM) and Cryo-TEM. Immunophenotyping of exosomes was measured by nano-flow cytometry, where the exosomes were labelled with antibodies targeting CD63, CD9 proteins. ECs were treated with these isolated exosomes for 24 hr and endothelial barrier integrity was determined by FITC-dextran permeability and trans-endothelial electrical resistance (TEER) from confluent endothelial monolayers. Cytokine production in ECs was measured by ELISA. In addition, tight junctional disruption was examined by immunofluorescent staining. Results: TMAO treatment enhanced both CD9 and CD63 protein levels in ECs as compared to control (n=5/group; p<0.05). Nanoparticle tracking analysis determined the particle size and revealed enhanced release of exosomes upon TMAO treatment in ECs. Additionally, TMAO-induced exosome secretion was confirmed by TEM analysis. EC-derived exosomes induced damage to the endothelial barrier integrity which was demonstrated by ECs permeability assay and TEER experiments. TMAO induced exosomal release also resulted in enhanced production of interleukin-1β in ECs (n=4/group; p<0.05). Immunofluorescence revealed that TMAO induced exosomal release markedly reduced tight junctional proteins such as ZO-1, ZO-2, and VE-cadherin (n=4/group; p<0.05). Conclusion: In conclusion, TMAO induced formation and release of exosomes from ECs both in vitro and in vivo. These activated EC-derived exosomes contribute to endothelial dysfunction and vascular injury. NIH R01HL148711 to Dr. Saisudha Koka This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.