Exosomes from Patients with Sickle Cell Disease and History of Acute Chest Syndrome Alter Endothelial Integrity In Vitro

Introduction: Acute Chest Syndrome (ACS) is the leading cause of death among children with sickle cell disease (SCD). While the phenotypic variability of ACS is incompletely understood, aberrant cell-cell interactions involving the endothelium are central to the pathophysiology. Recent studies suggest that circulating cell-derived extracellular vesicles, termed exosomes, can regulate endothelial integrity. We and others recently demonstrated that exosomes from SCD patients differentially affect endothelial integrity in vitro, but the relationship to specific complications of SCD, such as ACS, are unknown. Given the critical role of the endothelium in ACS, we hypothesized that exosomes from patients with a history of ACS induce increased endothelial damage compared to those without a previous ACS episodes.

Methods: Plasma was isolated from 33 patients with SCD. Patients were >4 weeks since transfusion and had no new health-related complaints. Control plasma samples were from children without SCD or known medical problems, who had a BMI < 95%ile. Exosomes were isolated from plasma using established methodologies. The cellular origin of exosomes was determined using Image Stream flow cytometry. To determine the effects on endothelium, exosomes were added to cultures of human microvascular endothelial cells (HMVEC-D). Intercellular junctions were visualized by immunofluorescent microscopy for VE-cadherin. To quantify effects on endothelial barrier integrity, HMVEC-D endothelial cells were grown to confluence on an Electric Cell-substrate Impedance Sensing (ECIS) array, treated with exosomes and then continuously monitored for 36 hours. Endothelial Nitric Oxide Synthase (eNOS) mRNA expression was assessed in HMVEC-D cells 24 hours post-exposure by qRT-PCR.

Results/Discussion: Flow cytometry demonstrated that the absolute exosome number was greatly increased in patients with SCD compared with controls. In contrast, no significant differences in total exosome numbers emerged between SCD patients based on ACS history. The origin of theexosomes was mainly erythroid (controls:9,661 ± 3,195 /100 uL vs. SCD: 31,338 ± 5,323 /100 uL, p<0.007), but significantly increased numbers of endothelial-, CD34+, lymphocyte-, and monocyte- derived exosomes were also detected. Although a minor population, ACS(+) patients had significantly more monocyte-derived exosomes than ACS(-) patients (monocyte exosomes ACS(-):45.89 ± 22.41 /100 uL vs. ACS(+): 477.4 ± 173.7 /100 uL, p=0.0218); exosomes from other sources did not differ. Immunolocalization of VE-cadherin showed that exosomes from SCD patients (especially ACS(+)) led to the formation of increased gaps between HMVECs as compared to untreated cells or cells treated with exosomes from control patients. ECIS recordings showed that samples from ACS(-) patients differed minimally from control patients, but exosomes from ACS(+)-patients greatly decreased monolayer resistance (relative resistance: ACS(+): 0.981±0.055 vs. ACS(-): 1.124±0.042; p = 0.006). Given its role in pulmonary endothelial dysfunction in ACS, we evaluated the ability of exosomes to induce eNOS expression. ACS(-) samples induced a ~50% increase in eNOS expression when compared to controls (controls;1.01± 0.00 versus ACS(-); 1.61 ± 0.06; p = 0.01) while ACS(+)-derived exosomes failed to induce any significant changes (ACS(+): 0.99 ± 0.05; p < 0.0001 vs. ACS(-)).

Conclusions: The results confirm and expand on our prior observations that circulating exosomes are increased in patients with SCD and contribute to the maintenance of endothelial integrity. Exosomes from ACS(+) patients disrupt the endothelial monolayer in vitro (unlike exosomes from SCD patients who never experienced ACS). We speculate that the increased exosomes contribute to the vascular pathology in all SCD patients. However, some SCD patients are protected by specific exosome cargoes, as in the ACS(-) patients whose exosomes induced eNOS expression by the cultured endothelial cells in vitro, which plays a critical role in endothelial health. Together, the current findings suggest that exosomes are differentially generated based on ACS history, and that their function/cargo (as opposed to their number), modulates endothelial function. Thus, SCD exosomes that induce endothelial dysfunction likely contribute to the pathophysiology of ACS, and may serve as risk-related biomarkers.