Abstract Background Pulmonary arterial hypertension (PAH) is characterized by progressive pulmonary vascular remodeling. Beyond established pathways, chronic inflammation and dysregulated innate immunity are now recognized as key drivers of disease. Neutrophil extracellular traps (NETs) have been directly implicated in propagating endothelial injury in PAH. However, the upstream cellular signals that promote sustained NET formation in the vascular lumen remain poorly defined. We hypothesized that extracellular vesicles (EVs), acting as circulating mediators of intercellular communication, could be a critical trigger for NETosis in this condition. Purpose This study aimed to determine whether EVs isolated from the plasma of patients with PAH are capable of inducing NETosis and to characterize their cellular origin. Methods Plasma was obtained from 36 patients with idiopathic PAH and 20 healthy controls. Circulating NET complexes (neutrophil elastase NE-DNA and myeloperoxidase MPO-DNA) were quantified by specific ELISAs. EVs were purified from platelet-poor plasma and characterized by high-sensitivity nanoparticle traccking analysis (NTA) and flow cytometry for neutrophil markers (CD45, CD66b) and phosphatidylserine (PS) exposure. EV morphology was assessed by transmission electron microscopy (TEM), and DNA content was quantified using a SYTOX Green assay. The functional capacity of purified EVs to induce NETosis was tested by incubating with neutrophils isolated from both PAH patients and healthy donors; NET formation was visualized and quantified via confocal microscopy after staining with Sytox Green. Results Plasma levels of NE-DNA and MPO-DNA complexes were significantly elevated in PAH patients compared to controls (NE-DNA: 0.79 ± 0.24 AU vs. 0.05 ± 0.0.04 AU, p0.001; MPO-DNA: 1.32 ± 0.74 AU vs. 0.38 ± 0.24 AU, p0.001). Purified PAH EVs were enriched in the neutrophil-specific markers CD45 and CD66b and exhibited high PS surface expression. TEM analysis revealed a subpopulation of PAH EVs with distinctive thread-like structures, and SYTOX staining confirmed they carried significantly more DNA than control EVs (p0.01). Functionally, PAH-derived EVs robustly induced NETosis in neutrophils from PAH patients, increasing the rate of SYTOX-positive NET-forming cells compared to unstimulated cells. Strikingly, the same PAH-EVs failed to trigger significant NETosis in neutrophils isolated from healthy donors. Conclusion(s) Circulating EVs in PAH carry a distinct neutrophil-derived signature, enriched in DNA and pro-inflammatory markers. These vesicles function as potent, disease-specific inducers of NETosis, selectively activating neutrophils from PAH patients but not from healthy controls. This peculiar functional role by pro-netotic EVs, establishes a novel mechanistic link between innate immune dysregulation and the sustained vascular inflammation that drives PAH pathophysiology thereby opens new avenues understanding PAH disease mechanism/s.
Khandagale et al. (Fri,) studied this question.