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Abstract P002: Aldosterone Promotes the Release of miRNA-Containing Exosomes from Endothelial Cells, Leading to Uptake by Smooth Muscle Cells


 Background:
 Aldosterone increases exocytosis from endothelial cells, which may contribute to the detrimental actions of aldosterone in the vascular system. We propose a role for exocytosis of exosomes; these small extracellular particles contain host cell genetic material and once released they can circulate in the blood and transfer their contents (e.g. miRNAs) to recipient cells. We aimed to examine the effect of aldosterone on exosome release from endothelial cells, the miRNA content of exosomes and finally, exosome trafficking of miRNA from endothelial cells to smooth muscle cells.
 
 
 Methods and Results:
 Exosomes were isolated via ultracentrifugation from the media of human coronary artery endothelial cells (HCAECs) following incubation with aldosterone (100nM) or vehicle (DMSO). Exosomes were analyzed using western blot analysis testing for exosome and cell markers, and by nanoparticle-tracking analysis (NanoSight); aldosterone increased exosome concentration by 2.65 fold (19.1x1010 particles/mL) compared to the vehicle control (7.2x1010 particles/mL). RNA was extracted from isolated exosomes and host HCAECs and levels of miR-24 measured by qRT-PCR. miR-24 levels were unaffected by aldosterone stimulation in HCAECs (0.58±0.19; p=0.07) but were significantly increased in exosomes isolated from the media (2.35±0.07 fold; p=0.03). To test exosome paracrine trafficking, HCAECs were transfected with a c.elegans miRNA (cel-miR-39) and incubated adjacent to human coronary artery smooth muscle cells (HCASMCs) separated by a 0.4 μm filter, with or without aldosterone. Levels of cel-miR-39 in HCASMCs increased significantly (2.42±0.52 fold; p=0.02) in aldosterone-stimulated cells, compared to control. Blocking transcription with Dactinomycin (2.07±0.61 fold; p>0.05) or blocking the mineralocorticoid receptor had no effect on aldosterone-mediated trafficking (3.43±1.49 fold; p>0.05).
 
 
 Conclusion:
 This study demonstrates that aldosterone: 1) increases exosome secretion from endothelial cells, 2) increases miR-24 content of exosomes and 3) contributes to an increased uptake of exosome-packaged microRNAs by smooth muscle cells. Together, this represents a novel mechanism by which aldosterone contributes to vascular disease.
