Circulating endothelial extracellular vesicles from sickle cell patients promote susceptibility of pulmonary endothelium to thrombotic injury

Abstract Introduction: Sickle cell disease (SCD) is a genetic hemoglobinopathy characterized by recurrent vaso-occlusion, chronic hemolysis, and systemic endothelial dysfunction, culminating in a pro-thrombotic and pro-inflammatory vascular milieu. The pulmonary endothelium, as a critical regulator of vascular homeostasis and barrier integrity, is particularly susceptible to dysregulation in SCD, contributing to severe complications such as pulmonary hypertension and acute chest syndrome. While endothelial activation is a hallmark of SCD, the role of endothelial extracellular vesicles (EVs), nano-sized membrane-bound vesicles secreted by endothelial cells, in modulating vascular responses remains unexplored. This study is the first to characterize endothelial EVs isolated from SCD patients and to assess their functional impact on pulmonary endothelial barrier integrity under thrombotic stress. Methods and Results: Endothelial EVs were isolated from the plasma of patients with SCD HbSβ⁰, HbSS, and HbSC, as well as from age-matched healthy controls, using CD31-targeted immunoenrichment. Transmission electron microscopy confirmed the presence of intact vesicles with preserved ultrastructure, consistent with canonical EV morphology across all groups. Nanoparticle tracking analysis revealed genotype-specific differences in size distribution. HbSS-derived EVs exhibited a significantly smaller mean diameter (128.4 ± 2.8 nm) compared to HbSβ⁰ (176.7 ± 8.2 nm), HbSC (178.0 ± 5.4 nm), and healthy controls (152.5 ± 7.1 nm). Western blot analysis confirmed consistent enrichment of the endothelial marker CD31 and the canonical EV marker CD63 across all SCD-derived endothelial EV preparations, with markedly elevated CD9 expression in EVs from HbSβ⁰ patient. To assess the impact of endothelial EVs on vascular function, human pulmonary microvascular endothelial cells (HPMECs) were exposed to EVs under basal and thrombin-stimulated conditions. Under basal conditions, EV treatment did not significantly alter transendothelial electrical resistance (TEER), indicating that the vesicles were not intrinsically cytotoxic or disruptive to barrier integrity. However, in the presence of thrombin (2U/mL), cells pre-treated with SCD-derived endothelial EVs exhibited a significant decrease in TEER compared to EVs from healthy controls, with the most pronounced effect observed following treatment with HbSS-derived EVs. This marked reduction suggests that HbSS-derived EVs strongly potentiate thrombin-induced endothelial dysfunction. In the absence of thrombin, HbSS-derived endothelial EVs also induced a marked increase in ICAM-1 and VCAM-1 expression in HPMECs, indicating direct activation of pro-inflammatory signaling pathways. This effect suggests the presence of bioactive cargo within HbSS EVs capable of stimulating endothelial cells independently of external inflammatory stimuli. The resulting upregulation of adhesion molecules is likely to enhance leukocyte recruitment and transendothelial migration, contributing to the microvascular occlusion observed during vaso-occlusive crises in HbSS patients. Conclusion Collectively, these findings demonstrate that endothelial EVs differ significantly in their biophysical properties, molecular composition, and functional impact depending on SCD subtypes. HbSS-derived endothelial EVs, in particular, display both the smallest size and the highest pro-inflammatory activity, suggesting a more active role in sensitizing the endothelium to inflammatory and thrombotic insults. These functional and structural differences are likely driven by underlying genotype-dependent alterations in EV biogenesis, membrane composition, or release mechanisms. The reduced size of HbSS-derived EVs may reflect the chronic oxidative stress, mechanical shear, and sustained endothelial injury characteristic of this genotype. In contrast, the relatively larger EVs observed in HbSβ⁰ and HbSC patients may result from alternative vesicle trafficking pathways or differences in endosomal maturation. Together, these patient-specific features support a model in which endothelial EVs contribute to the pathophysiological heterogeneity of SCD and may serve as valuable biomarkers or therapeutic targets for vascular dysfunction.