Exosomal Cargo Properties, Endothelial Function and Treatment of Obesity Hypoventilation Syndrome: A Proof of Concept Study

Abstract

Study objectives: Longitudinal studies support the usage of positive airway pressure (PAP) therapy in treating obstructive sleep apnea (OSA) to improve cardiovascular disease. However, the anticipated benefit is not ubiquitous. In this study, we elucidate whether PAP therapy leads to immediate improvements on endothelial function, a subclinical marker of cardiovascular status, by examining the effect of circulating exosomes, isolated from patients before and after PAP therapy, on naive endothelial cells.

Methods: We isolated plasma-derived circulating exosomes from 12 patients with severe OSA and obesity hypoventilation syndrome (OHS) before and after 6 weeks of PAP therapy, and examined their effect on cultured endothelial cells using several in vitro reporter assays.

Results: We found that circulating exosomes contributed to the induction and propagation of OSA/OHS-related endothelial dysfunction (ie, increased permeability and disruption of tight junctions along with increased adhesion molecule expression, and reduced endothelial nitric oxide synthase expression), and promoted increased monocyte adherence. Further, when comparing exosomes isolated before and after PAP therapy, the disturbances in endothelial cell function were attenuated with treatment, including an overall cumulative decrease in endothelial permeability in all 12 subjects by 10.8% (P = .035), as well as detection of a subset of 4 differentially expressed exosomal miRNAs, even in the absence of parallel changes in systemic blood pressure or metabolic function.

Conclusions: Circulating exosomes facilitate important intercellular signals that modify endothelial phenotype, and thus emerge as potential fundamental contributors in the context of OSA/OHS-related endothelial dysfunction. Exosomes may not only provide candidate biomarkers, but are also a likely and plausible mechanism toward OSA/OHS-induced cardiovascular disease.

Clinical trial registration: Registry: ClinicalTrials.gov, Title: AVAPS-AE Efficacy Study, URL: https://ichgcp.net/clinical-trials-registry/NCT01368614, Identifier: NCT01368614.

Keywords: cardiovascular disease; endothelial function; exosomes; obstructive sleep apnea; positive airway pressure.

© 2018 American Academy of Sleep Medicine.

Figures

Figure 1. Exosome-mediated in vitro effects on endothelial cell monolayer resistance and membrane tight junction proteins.

(A) Ensemble-averaged curves of ECIS measured endothelial cell barrier resistance changes over time after administration of exosomes from adult patients with severe OSA/OHS before (Pre-PAP; red line) and after (Post-PAP; green line) positive airway pressure therapy compared to endothelial cells incubated with plasma free media and empty exosomes (control; black line). (The y axis refers to a ratio of ECIS values to baseline ECIS values at start of experiment). (B) Evaluation of ECIS measured endothelial cell barrier resistance changes of all 12 individual patients (pt) comparing the effects on endothelial cells 24 hours after exosome administration. ECIS changes were evaluated before (Pre-PAP) and after 6 weeks positive airway pressure therapy (Post-PAP). The overall cumulative improvement in endothelial cell monolayer resistance for all 12 subjects was +10.8% (P = .035). (C) Effect of plasma exosomes on tight junction and membrane structure in naive endothelial cells. Representative images of at least six separate experiments illustrate exosome-induced changes in expression of VE-cadherin and zonula occludens (ZO)-1 expression patterns. The scale bars for all the representative images are 25 μm. Left figures represent before and right figures represent after PAP therapy. ECIS = electric cell-substrate impedance sensing, OHS = obesity hypoventilation syndrome, OSA = obstructive sleep apnea, PAP = positive airway pressure.

Figure 2. Exosome-mediated effects on endothelial cell expression of adhesion molecules, monocyte attachment of endothelial cells and changes in mRNA expression.

(A) Effect of plasma exosomes on adhesion molecule expression in naive endothelial cells. Representative images of at least six separate experiments show exosome-induced changes in expression of vascular cellular adhesion molecule (VCAM) and intercellular-adhesion molecule 1 (ICAM) with visually apparent reduction in post-PAP. (B) Effect of plasma exosomes on attachment of red fluorescent protein tagged monocytes. Representative images are shown for at least six separate experiments in different subjects. The scale bars for all the representative images are 25 μm. Left figures represent before and right figures represent after PAP therapy. (C) Quantification of monocyte attachment was performed by measuring immunofluorescence intensity of endothelial cells inoculated with exosomes from six subjects before (Pre PAP) and after 6 weeks of positive airway pressure therapy (Post PAP), and compared to plasma free media with empty exosomes (control). The net reduction in monocyte attachment from to Pre PAP to Post PAP was statistically significant (P = .0423). (D) Changes in mRNA fold changes of ICAM-1, VCAM-1, β-integrin, and eNOS expression in endothelial cells treated with either pre-PAP or post-PAP exosomes. PAP therapy was associated with significant reductions in the expression of both ICAM-1 and VCAM-1, along with significant increases in eNOS mRNA expression (P < .05 for all comparisons). eNOS = endothelial nitric oxide synthase, PAP = positive airway pressure.

Figure 3. Differentially expressed miRNAs in exosomal cargo in subjects pretreatment versus posttreatment (n = 8).