Endothelial-derived extracellular vesicles impair human pulmonary microvascular cell function in an in vitro model of sepsis-induced acute lung injury.

Acute respiratory distress syndrome (ARDS) remains a critical condition associated with high morbidity and mortality, particularly when triggered by sepsis. Endothelial dysfunction is a central hallmark of ARDS pathology, but the precise mechanisms underlying pulmonary microvascular dysfunction remain poorly understood. Extracellular vesicles (EVs) have emerged as crucial mediators of cell-cell communication during inflammation; however, their role in endothelial dysfunction in ARDS is less clearly defined. We utilized a human pulmonary microvascular endothelial cell (HPMEC)-based model of sepsis-induced acute lung injury to investigate whether inflammatory EVs (iEVs), derived from endothelial cells treated with bacterial lipopolysaccharide (LPS), impair naïve HPMEC function. EVs were characterized by nanoparticle tracking analysis, transmission electron microscopy, and immunofluorescence, confirming purity and uptake. iEV exposure significantly reduced barrier integrity by electric cell-substrate impedance sensing (ECIS) and increased cell migration; effects partially reversed by the toll-like receptor 4 (TLR4) inhibitor TAK-242. Adhesion and tube formation were unaffected. Pretreatment of donor HPMECs with the neutral sphingomyelinase inhibitor GW4869 attenuated the barrier-disrupting capacity of the resulting iEVs, implicating ceramide-dependent EV biogenesis in generating pathogenic cargo. Trypan Blue staining confirmed that these effects reflect altered signaling rather than cell death. iEV exposure upregulated TLR4, MyD88, interleukin-6 (IL-6), intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), E-selectin, and Jag1 mRNA, with TAK-242 attenuating IL-6 and ICAM-1 induction. Our results highlight endothelial-derived EVs and TLR4-dependent pathways as amplifiers of pulmonary vascular injury in sepsis-induced ARDS, identifying EV biogenesis and EV-mediated signaling as novel therapeutic targets.NEW & NOTEWORTHY EVs released from LPS-stimulated human pulmonary microvascular endothelial cells compromise barrier integrity and enhance migration via TLR4-mediated signaling. Inhibition of ceramide-dependent EV biogenesis with GW4869 attenuated barrier dysfunction, implicating specific EV subpopulations as pathogenic mediators. EV-TLR4 activation upregulates MyD88 and IL-6, suggesting an autocrine loop amplifying vascular injury in sepsis-induced ARDS, and identifying EV biogenesis and EV-mediated TLR4 signaling as novel therapeutic targets.